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Analysis of in vivo Glycine transporter 1 functions by
transgenic approaches
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim Fachbereich 14
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main
von
Deepti Lall
aus Delhi (India)
Frankfurt 2011
(D30)
Die vorliegende Arbeit wurde in der Abteilung Neurochemie am Max-Planck Institut für
Hirnforschung in Frankfurt am Main unter Anleitung von Prof. Heinrich Betz durchgeführt
und vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-
Universität in Frankfurt am Main als Dissertation angenommen.
Dekan: Prof. Dr. Dieter Steinhilber
1. Gutachter: Prof. Dr. Alexander Gottschalk
2. Gutachter: Prof. Dr. Heinrich Betz
Datum der Disputation:
A father's goodness is higher than the mountain,
A mother's goodness deeper than the sea.
For Mumma and Papa
INTRODUCTION
1 INTRODUCTION
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INTRODUCTION
1.1 Inhibitory neurotransmission in the CNS
Communication between the neurons is a chemical process. When a neuron is
stimulated, the electrical signal (action potential) travels down the axon to the axon
terminals and triggers a series of chemical changes. Upon stimulation, there is an influx of
the Ca++ ions into the neuron which initiates the release of the neurotransmitters. These
neurotransmitters can then fulfill different functions in the brain, mainly either excitation
or inhibition of the postsynaptic neuron. Excitatory neurotransmitters act to stimulate
the postsynaptic neuron whereas Inhibitory neurotransmitters tend to block the changes
that cause an action potential to be generated in the responding cell.
GABA and Glycine are the major rapidly acting inhibitory neurotransmitters in the
brain. GABA is ubiquitously present in the CNS and therefore GABAergic inhibition is the
most common form of inhibition in brain. Glycine, in contrast is the major inhibitory
neurotransmitter in the caudal regions of the brain especially in spinal cord and brain stem
where it is crucial for regulation of motorneuron activity. Like GABA, glycine also inhibits
neuronal firing by gating Cl- channels but with a characteristically different pharmacology.
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INTRODUCTION
The loxP sites are palindromic except for a 8-bp asymmetric core sequence that
provides each loxP site with an orientation (Hoess et al., 1986). If two loxP sites lie in the
same orientation on the same DNA strand, cre recombinase will catalyze the recombination
between the loxP sites and thus lead to a specific deletion of the flanked DNA segment. (Fig.
3.2).
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Fig.3.3: Role of Cre recombinaseCre recombinase, often abbreviated to Cre, is a type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites (red triangles) (B). When Cre recombinase (red circle) is introduced, either as a transgene by crossing into a mouse line carrying the targeted gene locus or on a viral vector, the DNA between the loxP sites (red triangles) is removed, thereby inactivating the gene (C).Adapted from Rosenthal and Brown, 2007.
MATERIALS AND METHODS
2 MATERIALS AND METHODS
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MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 Animals, Cell lines and Bacterial strains
Table 2-1: List of strains used in the study
Strain Source
Mouse (Mus musculus, C57/Black6) Charles River, Sulzfeld, Germany
Synapsin cre mouse line Zhu et. al. (2001)
GFAP cre mouse line Marino et. al (2000)
Emx cre mouse line Slezak et. al (2008)
Escherichia coli XL1-Blue StratageneTM (Amsterdam, Netherland)
E. coli DH5- InvitrogenTM (Carlsbad, USA)
Sv129/OlaHasd (E14TG2A) ES cell line Ulrike Mueller, Max Planck Institute for Brain Research, Frankfurt.
MTK-neo CD1 transgenic mice ref
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MATERIALS AND METHODS
2.1.2 Chemicals and plastic materials
All chemicals, unless otherwise stated, were ordered from the following companies:
Applichem (Darmstadt, Germany), Bio-Rad (Munich, Germany), Biotrend (Cologne,
Germany), Calbiochem Merck Biosciences (Schwalbach, Germany), Difco Laboratories
(Detroit, USA), Eppendorf (Hamburg, Germany), Fluka (Buchs, Switzerland), GE Healthcare
Biosciences (Freiburg, Germany), Gibco-BRL (Karlsruhe, Germany), Invitrogen (Carlsbad,
USA), Merck (Darmstadt, Germany), New England Biolabs (Ipswich, USA), Roche
Diagnostics (Mannheim, Germany), Roth (Karlsruhe, Gemany), Serva (Heidelberg,
Germany) and Sigma-Aldrich (Munich, Germany). All solutions were prepared with Milli-Q
water (Millipore, Wartford, USA).
All plastic materials were ordered from the following companies: Falcon (Le Pont De Claix,
France), Perbio Sciences (Bonn, Germany), Roth (Karlsruhe, Germany), Greiner (Darmstadt,
Germany), and Eppendorf (Hamburg, Germany).
2.1.3 Enzymes & others
All restriction enzymes were purchased from New England Biolabs, (Frankfurt am
Main, Germany) and Roche (Mannheim, Germany). The PAN Script DNA polymerase used in
PCR reactions was obtained from PANTM Biotech GmbH, (Aidenbach, Germany). The
Hotstart polymerase for genotyping inGlyT1 transgenic mouse tails was obtained from
Qiagen (Hilden, Germany). Ampli Gold taq polymerase from Applied Biosystems
(Darmstadt, Germany) was used for genotyping Glycine transporter 1 knockout mice, T4
DNA Ligase was obtained from Fermentas GmbH (St. Leon-Rot, Germany) and Proteinase K
and protease inhibitor cocktail were obtained from Roche (Mannheim, Germany).
2.1.4 DNA standard
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MATERIALS AND METHODS
As a reference marker for DNA, the Smart Ladder-Marker from Eurogentec (Cologne,
Germany) was used according to manufacturer’s instructions. This standard contains DNA
fragments of the following sizes: 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.8, 0.6, 0.4 and 0.2 kbp.
2.1.5 Protein standard
As reference for protein gels the SeeBlue2-Marker from Invitrogen (Carlsbad, USA)
was used according to manufacturer’s instructions. This marker contains the following
proteins: Myosin (250 kDa), Phosphorylase (148 kDa), BSA (98 kDa), Glutamate
Dehydrogenase (64 kDa), Alcohol Dehydrogenase (50 kDa), Carbonic Anhydrase (36 kDa),
Myoglobin Red (22 kDa), Lysozyme (16 kDa), Aprotinin (6 kDa) and Insulin (-chain) (4
kDa).
2.1.6 Membranes and films
Nitrocellulose membranes with a pore size of 0.45 µm from GE Healthcare were used
for southern blot analysis. For neutral transfer, Hybond-N+ and for alkaline transfer
Hybond-XL membranes were used. For Western blot analysis, nitrocellulose membrane
from GE Healthcare and ProtranTM from Schleicher and Schuell GmbH (Dassel, Germany)
were used. Autoradiographic films were purchased from BIOMAX MR (Kodak, Cedex,
France) or HyperfilmTM MP (A. Hartenstein, Erlangen, Germany) and GE Healthcare.
2.1.7 Commercial Kits for molecular biology
For standard molecular biology experiments, commercial kits were employed and all
the protocols were performed according to the manufacturer’s instructions.
Table 2-2: Commonly used kits.
Name Description Company
Plasmid kits Purification of plasmid DNA Qiagen, (Hilden, Germany)
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MATERIALS AND METHODS
(Mini, Midi, Maxi)
QIAquick Gel extraction kit Extraction of DNA from agarose gels
Qiagen
PCR purification kit Purification of PCR products Qiagen
SuperSignal Western Blotting chemiluminescent substrate kit
Enhanced chemilumescent (ECL) substrate for horseradish peroxidase (HRP) enzyme that permits low picogram detection of proteins in Western blot applications.
Pierce Thermo Scientific, (Schwerte, Germany)
QuickHyb Rapid hybridization solution
Used for quick hybridization of the radio-labeled probe to the nitrocellulose membrane
Stratagene,TM
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MATERIALS AND METHODS
2.1.8 Culture media and solutions
The following list of culture media and solutions were used during various cell-
biological and biochemical studies.
2.1.8.1 Bacterial Culture medium
Table 2-3: Culture medium used for growing bacteria
Name Composition
LB medium 1 % (w/v) NaCl, pH 7.5, 1 % (w/v) peptone , 0.5 %
(w/v) yeast extract
LB Agar LB medium with 1.5 % (w/v) agar
The LB or selection media were prepared by adding antibiotics in the required
concentrations to the autoclaved media, after it had cooled down to a temperature of ~55
°C. The final concentrations of the different antibiotics are listed in 2.1.8.2.
2.1.8.2 Antibiotics
Table 2-4: Antibiotics used for bacterial culture
Antibiotic Stock solution Final concentration
Ampicillin 100 mg/ml in water 50 µg/ml
Kanamycin 50 mg/ml in water 50 µg/ml
Tetracycline 10 mg/ml in 50 % ethanol 12.5 µg/ml
All antibiotics were sterile-filtered and stored at -20 °C.
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MATERIALS AND METHODS
2.1.8.3 Cell culture media for mammalian cells
Table 2-5: Cell culture media
Name Composition
DMEM medium +++
10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/ g/ml)μ , 2 mM L-Glutamine in 500 ml DMEM (Invitrogen).
Freezing medium 500 ml DMEM medium +++, 10 % (v/v) DMSO
Gelatin 0.5 % Gelatin in sterile distilled H2O
100 X poly-D-ornithine
150 g/ml, Poly-D-ornithine-hydrobromide in sterile distilled Hμ 2O
100 X poly-D-lysine
250 µg/ml Poly-D Lysine in distilled H2O
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MATERIALS AND METHODS
2.1.9 Medium and supplements for ES and MEF cell culture
Table 2-6: Cell culture media for ES and MEF cells
Name Composition Company
MEF medium DMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep stock (5000 U/µg/ml), 2 mM L-Glutamine
DMEM and FCS Gibco-BRL
2X MEF freezing medium
MEF medium, 20 % (v/v) DMSO
ES cell medium DMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/µg/ml), 2 mM L-Glutamine,10 mM NEAA, 0.1 mM -Mercaptoethanol, 1000 U/ml LIFβ
LIF (10,000 U/ml) :Life Technologies, (New York, USA).
ES cell selection medium
ES cell medium, 1000 U/ml LIF, 0.2 mg/ml Geneticin
Geneticin :Invitrogen
ES cell freezing medium
ES cell medium, 50 % (v/v) DMSO
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MATERIALS AND METHODS
All the media were sterilized through filtration with a 0.22 µm Bottle Top Filter and stored
for no longer than 2 weeks at 4 °C.
2.1.10 Electroporation of ES cells
For the electroporation of the ES cells the following media was used.
Table 2-7: Electroporation medium for the embryonic stem cells
Name Composition
10X HBSS 1X
1 M HEPES 20 mM
-Mercaptoethanolβ 0.1 M
1M NaOH 1 mM
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MATERIALS AND METHODS
The electroporation medium was prepared fresh for each round of electroporation and
sterilized through filtration with a 0.22 µm Bottle Top Filter (Becton Dickinson).
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MATERIALS AND METHODS
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MATERIALS AND METHODS
2.1.11 General buffers and solutions
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MATERIALS AND METHODS
All solutions were prepared in milliQ water from Millipore or as indicated. The
solutions were either sterile-filtered with a 0.45 µm Bottle Top Filter or autoclaved.
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MATERIALS AND METHODS
Table 2-8: Solutions for molecular biology
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MATERIALS AND METHODS
Page | 22
Name Composition
10X PBS 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, pH 7.3
PMSF 100 mM in isopropanol
10X DNA loading buffer 20 % (v/v) Ficoll 400, 0.1 mM EDTA, 1 % (w/v) SDS, 0,25 % (w/v) Bromophenolblue, 0,25 % (w/v) Xylene Cyanol FF
20X SSC buffer 3.0 M NaCl, 0.3 M Trisodium citrate pH 7.0
50X Denhardt’s solution 1 g BSA, 1 g PVP, 1 g Ficoll
50X TAE 242 g Tris base, 57.1 ml glacial acetic acid, 37.2 g Na2EDTA.2H2O, pH∼8.5
Brain P2 prep membrane preparation medium
0.33 M sucrose, 1 mM EDTA, 1 mM PMSF, 10 mM HEPES-Tris pH 7.4
DNA extraction “salting out” buffer 200 mM NaCl, 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 210 µg/ml Proteinase K (25 µg/µl stock solution)
DNA isolation Buffer PI 50 mM Tris-Cl, 10 mM EDTA pH 8.0, RNase A (100 µg/ml)
DNA isolation Buffer P2 200 mM NaOH, 1 %(w/v) SDS
DNA isolation Buffer P3 3 M Potassium acetate, pH 5.5
HEK293 cell lysis buffer 150 mM NaCl, 5 mM EDTA, 1 % (v/v) Triton-X 100, 0.25 % sodium deoxycholate, 0.1 % SDS, 50 mM HEPES-Tris pH 7.4, 1 tablet of Protease inhibitor complete (Roche Biosciences).
Krebs Henseleit medium 125 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.3 mM MgSO4, 10 mM Glucose, 25 mM HEPES-Tris pH 7.4
LacZ stain washing buffer 2 mM MgCl2, 0.01 % (w/v) sodium deoxycholate, 0.02 % (v/v) NP-40 in 1X PBS pH 7.3
LacZ staining buffer 5 mM Potassiumferrocyanide, 5 mM Potassiumferricyanide, 0.5 mg/ml X-Gal (in DMF) in 1X PBS pH 7.3
LacZ fixative for embryo staining 1 % formaldehyde, 0.2 % glutaraldehyde, 5 mM EGTA, 0.02 % NP-40 in 1X PBS
LacZ staining buffer for embryo staining
5 mM Potassiumferrocyanide, 5 mM Potassiumferricyanide, 2 mM MgCl2, 01 % Na deoxycholate, 0.02 % NP-40, 1 mg/ml X-Gal (in DMF) in 1X PBS pH 7.3
Lysis buffer ES cells for PCR 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 0.2 M NaCl
Lysis buffer for ES cells 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 200 mM NaCl, 62.5 µg/ml Proteinase K
MATERIALS AND METHODS
2.1.12 Solutions for protein biochemistry
Table 2-9: Composition of solutions
2.1.13 Solutions for immunocytochemistry and immunohistochemistry
Table: 2.10: Composition of solutions
Name Composition
2-4% PFA solution Add 2-4 g of PFA to 50 ml of sterile distilled water. Add a few drops of 1 N NaOH and heat at 55 °C till PFA dissolves. Add 10 mL of 10X PBS and make up the final volume to 100 ml with sterile distilled H2O.
Blocking and permeabilisation solution 1 % (v/v) goat serum, 2.5 % BSA, 0.1-0.3 % Triton-X 100 in 1X PBS pH 7.5
Quenching solution 100 mM glycine in 1X PBS pH 7.5
Quenching solution for DAB staining 0.3-1 % H2O2 in 1X PBS pH 7.5
Page | 23
Name Composition
10X Tris-Glycine buffer 30.2 g Tris base, 144 g glycine
4X Laemmeli buffer 8-11 % (w/v) SDS, 4 % (v/v) glycerin, 0.04 % (w/v) Pyronin Y, 20 % (v/v) -mercaptoethanol, 250 mMβ Tris-Cl pH 6.8
Blocking solution for western blotting
5 % (w/v) skim milk powder, 0.05 % (v/v) Tween-20 in 1X PBS
Ponceau-staining solution 3 % (w/v) Glacial acetic acid, 0.3 % (w/v) Ponceau S
SDS PAGE running buffer 1X TG buffer, 0.1 % SDS, pH 8.8
Stripping buffer for Western blot 420 µl -mercaptoethanol added to 15 ml Tris-Cl pH 6.8, 24 ml SDS (10 %) (v/v), 81 ml milliQ water
Western blot transfer buffer 20 % Methanol, 0.01 % SDS in 1X TG buffer, pH 8.8
MATERIALS AND METHODS
2.1.14 Vectors
Table 2-11: List of vectors used (in the study)
Name Origin/ Reference Antibiotic resistance
pBluescript II SK (+) Stratagene (Heidelberg, Germany)
Ampicillin
pBK CMV Stratagene (Heidelberg, Germany)
Kanamycin
pcDNA3.1 Invitrogen, (Carlsbad, USA) Ampicillin
pcDNA3.1/ DLC myc Petra Scholze, MPIH, Frankfurt Ampicillin
pCCALL2-IRES-EGFP/anton Lobe et.al. (1999) Ampicillin
pCCALL2-IRES-EGFP/anton/GlyT1
Chigusa Shimizu Okabe (this study)
Ampicillin
pEGFP-C3 Clontech (Heidelberg, Germany)
Ampicillin
pPGKcrebpA Klaus Rajewsky, (Harvard Medical School, Boston, MA, U.S.A.)
Ampicillin
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MATERIALS AND METHODS
2.1.15 Antibodies
Table 2-12: List of primary antibodies used (in the study)
Name Antigen Species Dilution
WB
ICC/IHC
Source
mAb4a hybridoma supernatant
AS 96-105 from GlyR 1α
mouse Dr. Carmen Villman, Uni-Erlangen
GFP GFP isolated directly from Aequorea victoria
rabbit 1:1000 1:500 Invitrogen
VIAAT AS 75-87 from VIAAT rabbit 1:1000 Synaptic Systems, Göttingen
myc 9106 EQKLISEENL rabbit 1:200 Abcam, Cambridge, U.K
-Gal 4761β Full length native protein (purified from E. coli).
rabbit 1:250 Abcam, Cambridge, U.K
GlyT1(M7/M14)
rabbit 1:1000 Eulenburg et.al., 2010
GlyT2N N terminal cytoplasmic domain of rat GlyT2
rabbit 1:2000 Gomeza et.al., 2003a
GRP75 (JG1) 2799
AS 615-633 of Mouse Grp75
mouse 1:1000 Abcam, Cambridge, U.K
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MATERIALS AND METHODS
Table 2-13: Fluorescent conjugated secondary antibodies used for ICC and IHC
Name Dilution Source/Reference
Alexa 488-anti-mouse 1:500 Molecular Probes (Eugene, USA)
Alexa 546-anti-mouse 1:500 Molecular Probes (Eugene, USA)
Alexa 488-anti-rabbit 1:500 Molecular Probes (Eugene, USA)
Alexa 546-anti-rabbit 1:500 Molecular Probes (Eugene, USA)
Table 2-14: Peroxidase-linked secondary antibodies for western blot
Name Dilution Source/Reference
HRP-anti-mouse 1:10000 Dianova (Hamburg, Germany)
HRP-anti-rabbit 1:10000 Dianova (Hamburg, Germany)
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MATERIALS AND METHODS
2.2 METHODS
2.2.1 Biochemistry and molecular biology methods
2.2.1.1 Growth conditions and culture methods for microbial culture
The microbial cultures were grown under standard conditions as described (Bertani,
1951; Rose, 1990; Sambrook et.al., 1989). The E. coli strains were grown O/N in liquid LB
containing the appropriate antibiotics at 37 °C on an orbital shaker at 180-200 rpm. The
final concentrations of different antibiotics that were used for growing the cells are
described in 2.1.8.2.
2.2.1.2 Preparation of E. coli chemical competent cells
E. coli XL1-Blue cells were spread on agar plates containing 10 µg/ml tetracycline
and incubated O/N at 37 °C. Next day, a single colony was inoculated in a pre-culture of 5 ml
LB medium and incubated O/N at 37 °C with constant shaking. On the following day, the
O/N culture was transferred to 500 ml of pre-warmed LB medium and incubated at 37 °C at
300 rpm until the OD600 reached 0.5-0.7. The bacteria were then harvested by∼
centrifugation at 2600 x g, for 5 min at 4 °C. The cell pellet was resuspended in 8 ml of
sterile ice-cold 50 mM CaCl2 solution and kept for 1 hr at 4 °C. The cells were centrifuged at
1000 x g, 5 min at 4 °C and the pellet was carefully resuspended in 8 ml of sterile ice-cold 50
mM CaCl2 solution containing 40 % (v/v) glycerol. 200 µl aliquots of the bacteria were
frozen in liquid nitrogen and stored at –80 °C.
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MATERIALS AND METHODS
2.2.1.3 Preparation of E. coli electrocompetent cells
30 ml LB-medium was inoculated with a single colony of E. coli XL1-Blue bacteria
and incubated O/N at 37 °C at 200 rpm. 10 ml of the O/N culture was transferred next day
to 1 l of pre-warmed LB medium and incubated at 37 °C and 300 rpm until the OD 600
reached 0.5-0.7. The bacteria were then kept at 4 °C for 10-15 min and afterwards were∼
harvested by centrifugation at 3500 rpm, for 15 min at 4 °C. From here all the steps were
performed on ice using pre-chilled sterile solutions and glassware. The cell pellet was
subjected to three successive washes (resuspension in ice-cold ddH2O and subsequent
centrifugation at 3500 rpm for 15 min at 4 °C). Subsequently, the pellet was washed once
with 10 % (v/v) glycerol, and finally resuspended in 5 ml of 10 % glycerol. 100 µl aliquots
of the bacteria were frozen in liquid nitrogen and stored at –80 °C.
2.2.1.4 Preparation of bacterial glycerol stocks
After growing the bacterial culture in LB medium supplemented with appropriate
antibiotics, till the OD600 reached 0.5-0.7∼ , 800 µl of cell suspension was mixed with 800 µl
of sterile glycerol. The glycerol stocks were aliquoted, directly frozen in liquid nitrogen and
stored at -80 °C till further use. For revival of bacterial culture, 4-5 ml of LB medium was
inoculated by tiny amount of glycerol stock using an inoculation loop or autoclaved
toothpick. The inoculated LB medium was incubated O/N at 37 °C with gentle shaking.
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MATERIALS AND METHODS
2.2.1.5 Heat shock transformation of plasmid DNA
The microcentrifuge tubes containing 100 µl of chemical competent cells were
thawed on ice for 5 min before transformation. 10-50 ng of DNA were added to the
chemical competent cells and incubated on ice for 30 min. The transformation mixture was
heat-pulsed at 42 °C for 45 seconds and placed on ice for 2-3 min. 900 µl of pre-heated LB
medium (2.1.8.1) was added and incubated at 37 °C for 1 hr on a shaker at 200-225 rpm.
After recovery the bacterial cells were then plated on LB agar plates (2.1.8.1) containing the
appropriate antibiotic and incubated O/N at 37 °C.
2.2.1.6 Electroporation
Shortly before transformation, the E. coli electrocompetent cells were thawed on ice
for 5 min. The DNA was mixed with the cells and incubated additionally on ice for 20 min.
The bacteria-DNA mixture was added to the electroporation cuvette and placed in the
electroporation chamber of “Gene PulserII” (Bio-Rad, Munich, Germany) and electroporated
with the following settings: voltage of 2.5 kV, pulse controller low resistance 200 Ω, and
capacitance 25 µF. The cells were collected into 1 ml LB medium (2.1.8.1) and incubated on
a shaker at 37 °C for 1 hr. The bacterial culture was then plated on solid LB agar plates
containing appropriate antibiotics and incubated at 37 °C O/N.
2.2.2 Isolation and purification of plasmid DNA
2.2.2.1 Small-scale purification of plasmid DNA
The small scale plasmid DNA preparations were carried out using Qiagen’s Plasmid
mini kit (QIAGEN, Hilden, Germany). The DNA preparation is carried out by a modified
alkaline lysis protocol (Birnboim and Doly, 1979) followed by binding of the DNA to an
anion-exchange resin under appropriate pH and low-salt conditions and subsequent
washing and elution steps.
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MATERIALS AND METHODS
In brief, 5 ml LB medium containing the appropriate antibiotic were inoculated with
transformed E. coli XL1-Blue bacteria and incubated O/N at 37 °C, 200 rpm. Cells were then
pelleted at 5000 x g for 15 min and plasmid purification was performed following the
manufacturer’s instructions. The bound plasmid DNA was eluted from anion-exchange
resin using 50 µl low TE and stored at -20 °C.
2.2.2.2 Large scale plasmid DNA preparations ("midi/maxi preps")
Preparative purification of plasmid DNA was carried out using Qiagen’s Plasmid midi
kit or Plasmid maxi kits. In brief, a volume of 100 or 250 ml LB medium containing the
appropriate antibiotic was inoculated with transformed E. coli XL1-Blue bacteria and
incubated O/N at 37 °C and 200 rpm. Cells were then pelleted at 5000 x g for 15 min and
plasmid purification was performed following the manufacturer’s instructions. The eluted
DNA was resuspended in water or low TE to a final concentration of 1 µg/µl and stored at -
20 °C.
2.2.2.3 DNA extraction from mouse tissue
For the extraction of DNA from mouse tissue, approximately 3 mm of tails of
newborn mice were digested in a solution containing 350-500 µl of mouse tail lysis buffer
(2.1.11). For the brain samples, different regions of the brain were directly frozen on dry ice
after isolating them from mice and 350-500 µl of lysis buffer (2.1.11) was added to the
samples. The samples were incubated O/N at 56 °C and 800 rpm.
2.2.2.4 Phenol-chloroform extraction of the genomic DNA
The tubes containing the O/N digested samples were vortexed briefly and
centrifuged at 13000 rpm for 5 min in a tabletop centrifuge. The supernatant was
transferred to a new tube and an equal amount of mixture of
phenol:chloroform:isoamylalcohol (25:24:1) was added. The tubes were vortexed briefly
and centrifuged at 13000 rpm for 5 min. The upper aqueous phase was transferred to a new
sterile eppendorf tube and an equal amount of chloroform:isoamylalcohol (24:1) was
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MATERIALS AND METHODS
added. The tubes were vortexed briefly again and centrifuged at 13000 rpm for 5 min. The
supernatant was collected in a new eppendorf tube and 500 µl of iso-propanol was added to
the sample. Tubes were shaken and centrifuged again for 10 min at 13000 rpm to
precipitate the genomic DNA. The supernatant was discarded. The pellet was washed with
250 µl of 75 % ethanol, air-dried and resuspended in 100 µl of low TE buffer (2.1.11).
2.2.2.5 Genomic DNA isolation by “salting out”
Alternatively, the genomic DNA was isolated by salting out extraction. The tails were
digested overnight at 56 °C in lysis buffer for ‘salting out extraction’ (2.1.11). Tubes were
vortexed briefly and centrifuged at 13000 rpm for 5 min in a tabletop centrifuge. The
supernatant was transferred to a new tube and 150 µl of saturated NaCl was added to it.
The tubes were then incubated at 100 °C for 20 min and subsequently on ice for 15 min.
The sample mix was centrifuged for 15 min at 13000 rpm in a table top centrifuge at room
temperature. The supernatant was transferred to a new tube and 850 µl of 100 % ethanol
was added. Tubes were shaken and centrifuged for 10 min at 13000 rpm to precipitate
genomic DNA. The supernatant was discarded. The pellet was washed with 500 µl of 75 %
ethanol, air-dried, and resuspended in 100 µl of low TE buffer (2.1.11).
2.2.3 Analysis of the purified DNA
2.2.3.1 Agarose gel electrophoresis for the separation of the DNA fragments
For the separation of DNA fragments agarose gels ranging from 0.7 % to 3.0% were
used. The desired amount of agarose was dissolved in 1X TAE buffer by heating in a
microwave. After the agarose had cooled, ethidiumbromide (1 g/ml) was added to it. Theμ
warm agarose was slowly poured into a gel tank and combs were inserted. The set was kept
undisturbed till the agarose polymerized.
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MATERIALS AND METHODS
Then 1X TAE buffer was poured slowly into the tank till the buffer level stands 0.5-0.8 cm
above the gel surface. Wells were formed by gently lifting the combs. Standard DNA marker
(2.1.4) and DNA samples (containing glycerol and tracking dye) were loaded into the well.
After loading, the set up was connected to power supply and voltage was set to 120 V (or 8
to 10 V/cm length). The gel was run until the tracking dye reached ¾ of the gel length or
until the desired separation was achieved. For documentation, snapshots of UV
transilluminated gels were taken.
2.2.3.2 Determination of DNA concentration by spectrophotometry
According to the Lambert-Beer law the absorption of an aqueous solution of a
substance is directly proportional to its concentration, A=ε*c*l, where ε is the molar
extinction coefficient (M-1cm-1), c the concentration (M) and l is the path length of the light
through the sample (cm). The absorption is measured at 260 nm and 280 nm, which
represent the absorption maxima for nucleic acids and proteins, respectively. The
measured absorption is the sum of the absorptions of all the bases in the solution. The
concentration of the DNA solution was analyzed using Nanodrop ND-100 (Thermo Fisher
Scientific, Waltham, USA). The purity of the DNA preparation was estimated by calculating
the ratio of absorbances at 260nm and 280nm (A260/A280).
For RNA: A260/A280=2.0,
DNA: A260/A280=1.8 and
Protein: A260/A280=0.6.
A ratio of 1.8 - 2.0 is desired when purifying nucleic acids. If the ratio is less than 1.7, the
solution is probably contaminated by protein or phenol. The concentration of plasmid DNA
was analyzed and adjusted to a concentration of 1 µg/µl (1 OD260 = 50 µg/µl).
2.2.3.3 Extraction of DNA from agarose gels
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MATERIALS AND METHODS
To isolate the desired DNA fragments from a sample after restriction digestion or
PCR amplification, it was subjected to a gel electrophoresis (2.3.2.9). Under 302 nm
illumination, fragments of the desired size were cut out of the gel using a sterile scalpel and
placed in 1.5 ml micro-centrifuge tubes. DNA was purified with the QIAquick Gel Extraction
Kit (2.1.7) based on the principles of spin-column technology with the selective binding of
the DNA to silica-gel membrane. The purified samples were vacuum-dried and resuspended
in 10 µl H20.
2.2.3.4 DNA-sequencing
The separation, detection, and sequencing, of DNA products were performed by the
service provider MWG Biotech. Inc. (Ebersberg, Germany).
2.2.4 Enzymatic modification and manipulation of the DNA
2.2.4.1 Restriction digest of the DNA
Restriction endonucleases are enzymes that recognize specific sequences within
dsDNA and cut both DNA strands. Smith and Nathans (1975) discovered and characterized
the restriction endonucleases that are commonly used in the molecular cloning. These
enzymes cleave at specific sites within their recognition sequence that ranges from 4-8 bp
in length and is in most cases palindromic. The hydrolysis of both dsDNA strands can
generate 5’- protruding, 3’-protruding or blunt ends. The 5’ ends are always
phosphorylated and the 3’ends are hydroxylated. These characteristics make them a useful
tool in molecular biology for sequence specific fragmentation of DNA. One unit of the
restriction enzyme is defined as the amount of enzyme required to cut 1 g of DNA in 1μ
hour at 37 °C. For analytical DNA digests, usually 500 ng-10 g (for southern blot) of theμ
DNA was digested with 1-10 units of the corresponding enzyme at 37 °C for 1-2 hr. For
double digests involving enzymes requiring incompatible buffers, the DNA was digested
sequentially. Restriction enzymes were then inactivated at 65 °C for 20 min (for heat
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MATERIALS AND METHODS
sensitive enzymes) and the DNA fragments were isolated after electrophoretic separation
in an agarose gel using QIAQuick gel extraction kit.
2.2.4.2 Dephosphorylation of the DNA digested with restriction
Terminal 5’-phosphoryl groups can be enzymatically removed by treating dsDNA
with calf intestinal phosphatase (CIP), thereby preventing unwanted re-ligation of
restriction digested DNA. One unit of CIP is defined as the amount of enzyme required to
hydrolyze 1 M p-nitrophenolphosphate to nitrophenol in 1 min at 37 °C. To removeμ
5’phosphoryl groups to prevent re-ligation, 1-2 U of CIP was added to a heat inactivated
'restriction digest' and incubated for 30 min at 37 °C. Since CIP cannot be heat inactivated,
the treated DNA was subsequently gel purified using QIAquick gel extraction kit, (2.1.7).
2.2.4.3 Ligation of DNA fragments
The bacteriophage T4-encoded enzyme 'DNA ligase' catalyses the formation of
phosphodiester bonds between neighboring 3’-hydroxyl- and 5’-phosphoryl-termini. It
requires Mg2+ ions and ATP as co-factors. The efficacy of a ligation reaction is influenced by
several factors, e.g. incubation temperature, reaction volume and the concentration of DNA
termini. Addition of polyethylene glycol in the ligation buffer enhances the ligation efficacy
and reduces the incubation time. The enzymatic activity is measured as the ‘cohesive’ end
ligation unit', and is defined as the amount of enzyme required to achieve a 50% ligation of
HindIII digested λ-DNA in 30 min at 16 °C in 20 l reaction volume and a 5’ terminiμ
concentration of 0.12 M (300 g/ml).μ μ
DNA fragments were ligated by mixing 25-50 ng vector DNA with a threefold excess of
insert DNA. 0.5 l of T4-ligase and 1 l of 10 X ligation buffer were added and the reactionμ μ
mixture was brought to a final volume of 10 l with ddHμ 2O. The reaction was incubated O/N
at 16 °C. The reaction mixture was used directly for transformation without any further
purification.
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MATERIALS AND METHODS
2.2.5 DNA amplification: Polymerase chain reaction
The polymerase chain reaction allows the in vitro amplification of a specific DNA
sequence (Mullis et al., 1986). DNA synthesis starts at two primers that are flanking the
sequence to be amplified. One of the primers anneals to the sense and the other one to the
anti-sense strand of the amplicon. DNA synthesis is carried out in a thermocycler shifting
temperatures between 95 °C for the initial denaturation of the template, the specific
annealing temperature of the primers and 72 °C for the synthesis reaction. The DNA
polymerase of the thermophilic bacterium Thermus aquaticus (Taq polymerase) is routinely
used for the PCR which catalyzes the synthesis reaction at a temperature of 72 °C The
reaction mixture consists of a DNA template, specific primers to amplify the amplicon,
dNTPs and Taq polymerase with corresponding salt and optimal pH conditions. Usually, 24-
30 cycles are necessary to obtain sufficient quantities of the PCR product for subsequent
steps. For current project, Taq™ DNA polymerase (Invitrogen); PAN scriptTM polymerase
and Qiagen hotstart polymerase were used for different PCR reactions as listed Appendix II.
The PCR conditions used for different PCR reactions are described in Appendix III.
2.2.6 Random-labeling of DNA probes
DNA probes were radioactively labeled with -[Pα 32]-dCTP using the Random
Primed DNA Labeling Kit (Roche Applied Sciences). In brief, 10 ng – 3 µg of the DNA to be
labeled were denatured by incubating at 95 °C for 5 min. The DNA was centrifuged and then
subsequently used for labeling.
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MATERIALS AND METHODS
Table 2-15: Reaction mix for random labeling of DNA probes
Component Amount
Template DNA 9 µl (10 ng – 3 µg)
dTTP 1 µl
dATP 1 µl
dGTP 1 µl
Hexanucleotide buffer 2 µl
-[Pα 32]-dCTP 5 µl
Klenow enzyme 1.5 µl
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MATERIALS AND METHODS
The reaction was thoroughly mixed by pipeting and then incubated at 37 °C for 1 hr. After 1
hr, the reaction was stopped by adding 30 µl of STE buffer (2.1.11). The reaction mix was
vortexed and purified using G-50 columns (GE Healthcare Biosciences Freiburg, Germany).
The labeled probe was added to the column and centrifuged at 3000 rpm for 2 min. 1 µl of
the radiolabeled probe was used for measuring the radioactive counts. The probe was then
heated at 95 °C and added to the membrane containing the hybridisation buffer and
incubated at 42 °C O/N.
2.2.6.1 Southern blot analysis of genomic DNA
10 µg of genomic DNA was fully cut overnight with appropriate restriction enzymes
in a final volume of 35 µl. The samples were separated on 0.8 % 1X TAE agarose gel at 1.5
V/cm for 7 hr. The gel was treated sequentially with denaturation solution and
neutralization solution (2.1.11) and then transferred using neutral transfer, O/N onto a
nylon membrane (2.1.6) in 10X SSC. After the transfer, the DNA was crosslinked to the
membrane by UV irradiation (StratageneTM, UV crosslinker) and stored at -20 °C until
further use.
Hybridization reactions were performed either with QuickHyb hybridization solution
(StratageneTM) or hybridization solution (Table 2-16) in an oven equilibrated to the
appropriate temperatures. Blots were exposed at –70 °C using Kodak Biomax MR film
(2.1.6) with intensifying screens and developed after 3 days
Table 2-16: Hybridisation solution for Southern blot
Component Amount
Formamide 20 ml
Salmon sperm DNA* 1 ml
1M NaH2PO4, pH 6.5 2 ml
10 % SDS 2 ml
50X Denhardt’s sol
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MATERIALS AND METHODS
4 ml
0.5 M EDTA, pH 8.0 0.4 ml
20X SSC 12 ml
*Salmon sperm DNA was heated at 95 °C for 2-5 min and then pre-mixed with formamide
solution. All the rest components were added later.
2.2.7 Brain total RNA preparation
Different regions of mice brains were isolated and directly frozen on dry ice. Total RNA
from the brain samples was extracted using peqGOLD RNA islolation solution (PEQLAB
Biotechnologie GMBH, Erlangen, Germany) according to manufacturer’s instructions.
2.2.7.1 First strand c-DNA synthesis
cDNA synthesis is based on the characteristic feature of eukaryotic messenger RNAs
to a harbor defined polyadenylated tail on the 3′ end. The cDNA was mainly synthesized for
RT-PCR. Total RNA was mixed with oligo-dT primers. The amplification reaction was
carried out by Reverse TranscriptaseTM (Roche) at 50 °C.
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MATERIALS AND METHODS
In brief, 50 pmol of oligo-dT primer and 1 g total RNA were added to a nuclease-freeμ
microfuge tube. The mixture was heated at 65 °C for 10 min. 4 l of 5 X First-Strand Buffer,μ
0.5 l of Protector RNase inhibitor, 2 l of 10 mM dNTP’s and 0.5 l of reverseμ μ μ
transcriptase were added to the tubes and final volume of the mix was adjusted to 20 lμ
with ddH2O. This mix was incubated in the thermocycler with the following settings: 25 °C
for 10 min, 50 °C for 60 min. Finally the reaction was terminated by heating the reaction
mix at 85 °C for 5 min. The cDNA was then used as template PCR amplification. To obtain
pure cDNA which is required for PCR amplification, the mixture was incubated with 1 l (2μ
units) of E. coli RNase H at 37 °C for 20 min to remove >1kb RNA.
2.2.8 Cell biology methods
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MATERIALS AND METHODS
2.2.8.1 Coating of coverslips and culture plates with poly-D-ornithine and poly-D-
lysine
To facilitate the attaching of cells on the surface, sterile and ethanol-washed
coverslips (placed in 24-well-plates) and 6-well plates were incubated for a minimum of 2
hours with poly-D-ornithine or poly-D-lysine (15 µg/ml in PBS, pH 7.5) (Sigma, USA) at 37
°C and 5 % CO2. Before use, the coating solution was removed and wells were washed three
times with 1X PBS.
2.2.8.2 Culture and maintenance of HEK 293T cells
All steps were performed in a sterile hood with sterile solutions and media (MEM or
DMEM+++). Solution and media were preheated 30 min before use in a water-bath at 37 °C.
Twice per week, cells with a confluence of approx. 70-90 % were splitted. For splitting, cells
were washed once with 4 ml PBS. Cells were detached from the surface by addition of 1 ml
of preheated 0.25 % trypsin/EDTA (Gibco BRL). Finally, cells were resuspended in 5 ml of
the medium and plated on new 10 cm dishes containing 10 ml of the suitable culture
medium (2.1.9). Cells were cultured in a waterlogged incubator at 37 °C and 5 % CO2. After
30 culture passages, a new batch of cells was thawed.
2.2.8.3 Freezing and thawing of cell lines
Cells growing in a 10 cm dish with a confluence of approx. 80-90 % were washed
once with PBS (pH 7.5) and incubated for 1 min with 1 ml of 0.25 % trypsin/EDTA. The
cells were centrifuged at 800 rpm and resuspended in the culture medium. 1 volume of 2X
freezing medium (2.1.8.3) was added to the suspension. The cell dilution was then
transferred in 1 ml aliquots to Cryo-TubesTM (Thermo Fischer Scientific) and the tubes were
left overnight at -80 °C in a cryocontainer filled with isopropanol. Cells were stored in liquid
N2 until further use.
To revitalize frozen cells, a very rapid warming up of the tube in a water bath at 37 °C was
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MATERIALS AND METHODS
carried out. The thawed aliquot was mixed with 10 ml of prewarmed culture medium
(2.1.8.3) and distributed in 10 cm culture dishes. The cells were grown and passaged as
explained above in 2.2.8.2.
2.2.8.4 Transfection of HEK 293T cells using Lipofectamine 2000TM
HEK 293T cells were seeded on poly-D-ornithine-coated coverslips with culture
medium in the absence of antibiotic. Lipofection was carried out following manufacturer’s
instructions.
Briefly, a day before transient transfection, HEK 293T cells were seeded in 24-well (30,000
cells/cover-slip) or 6-well plates (50,000 cells per well) in antibiotic-free cell culture media
(2.1.8.2) On the next day for transfection of individual cover-slips, 2 µl of Lipofectamine
2000TM and 0.8 µg of plasmid DNA were separately incubated in 50 µl of DMEM medium for
5 min. For a 6-well plate, 4 µg of DNA with 10 µl of lipofectamine was used. After 5 min, the
solutions containing the DNA and lipofectamine were mixed and incubated at room
temperature for 20 min. Next the plasmid DNA with lipofectamine containing DMEM
medium was added directly onto the wells. For double transfection, 0.4 µg of the respective
plasmids (2 µg for a 6-well plate) were used for the same amount of the reaction mix as
described above. After 4 hr, culture dishes with transfected cells were supplemented with
500 µl or 10 ml of DMEM containing serum and antibiotics and further incubated for 12-24
hr at 37 °C and 5 % CO2 to allow the expression of the transfected constructs.
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MATERIALS AND METHODS
2.2.8.5 Preparation of lysates from HEK 293T cells
After 24-hr expression by HEK 293T cells, the cells were washed twice with PBS and
500 µl of HEK 293T cell lysis medium (2.1.11) was added to the plates. The cells were
scraped from the bottom of the culture plate using a cell scraper and transferred to a sterile
1.5 ml eppendorf tube. Finally, cell lysate debris was removed by centrifugation for 18000 x
g for 15 min. The supernatant was directly used for analysis or stored at -20 °C.
2.2.8.6 Immunocytochemistry on HEK 293T cells
Cells cultured on cover-slips coated with poly-D-ornithine or poly-D-lysine in 24-
well plates were washed once with ice-cold PBS and incubated with 0.5 ml of ice-cold 4 %
PFA solution (2.1.13) for 10 min on ice. The cells were then washed thrice with ice-cold
PBS. To quench unspecific fluorescence due to fixative procedures, cells were incubated for
10 min with 100 mM glycine in PBS followed by three subsequent washes with PBS. To
permeabilize cell membranes and block unspecific binding of the primary antibody, the
cells were incubated in 0.5 ml of blocking solution (2.1.13) for 1 hr at RT. Subsequently
incubation with the primary antibody was done (diluted in blocking solution, appropriate
dilutions are summarized in 2.1.15). 30 µl of the dilution was loaded onto a piece of
Parafilm (Pechiney, Chicago, USA) in a moist chamber to prevent drying of the solution.
Coverslips were then placed with cells facing down over the drop and incubated overnight
at 4 °C. On the next day, coverslips were washed with PBS for 10 min each. The cells were
then incubated with a fluorophore-linked secondary antibody diluted in blocking solution
(2.1.15) for 45 min at room temperature. The cover-slips were then washed thrice with
PBS, 10 min each. For the staining of cell nuclei, cells were incubated in DAPI solution
diluted 1:1500 in PBS for 10 min at RT in a dark chamber and again washed twice with PBS.
Cover-slips were mounted on microscope glass slides (76 x 26x 1 mm) (Paul Marienfeld,
Lauda-Königshofen, Germany) by placing them on a drop of Aqua Polymount (Polysciences
Inc. Warrington, USA) upside down. They were allowed to dry and stored at 4 °C until
further visualized.
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MATERIALS AND METHODS
2.2.8.7 Cryostat brain sectioning
For immunohistochemical analysis, mice were anesthetized by inhalation of
isofluoran (Deltaselect, Pfullingen, Germany) and killed by cervical dislocation. The head
was cut off with scissors; brain was carefully removed from the skull and rapidly frozen on
dry ice. The frozen brain was then embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude,
Netherlands), placed on a cryostat holder and left at -18 °C to allow the embedding gel to
embed the tissue. Transverse or sagittal slices of 12-20 µm width were cut in a Cryostat
(Leica, Jung Frigocut, 2800E). 4-6 slices were transferred to coated microscope glass slides
(25x75x1.0 mm, SuperFrostR Plus, Menzel Glaeser, Braunschweig, Germany) and either
processed rapidly or stored for further analysis at -80 °C.
For checking EGFP expression, the brain was dissected out from the skull and washed
briefly with ice-cold PBS and pre-fixed in freshly prepared ice-cold PFA solution (2.1.13) at
4 °C overnight. The following day, pre-fixed brains were washed with ice-cold PBS for 15
min at room temperature. For cryoprotection, brain samples were transferred to a solution
of 10 % sucrose/ PBS and incubated for 4 hr at 4 °C. Next, they were transferred to 20 %
sucrose/ PBS and incubated O/N 4 °C. The following day, the solution was change to 30 %
sucrose/ PBS and brains additionally incubated O/N 4 °C. The brain was then dried
completely to exclude out any solvent. The brain were then embedded in Tissue-Tek
(Sakura Finetek, Zoeterwoude, Netherlands) and incubated on ice to allow the medium to
penetrate into the tissue. Finally, the embedded brains were rapidly frozen on dry ice and
stored at -80 °C till further use.
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MATERIALS AND METHODS
2.2.8.8 Immunostaining on brain sections
For immunostaining, tissue slides were dried at room temperature and fixed for 10
min in ice-cold 4 % PFA (2.1.13). Afterwards, slides were washed thrice for 10 min with
PBS to completely remove the fixative. Slides were then incubated in quenching solution
(2.1.13) for 10 min at room temperature. Membrane permeabilisation and unspecific
binding of the antibody was blocked through the application of blocking solution (2.1.13)
for 2 hr at room temperature. The slides were incubated in primary antibody (appropriate
dilutions in 2.1.15 in blocking solution) O/N at 4 °C in a moist chamber. Next day, unbound
antibody was washed out by washing thrice with PBS for 10 min each. The slides were
incubated with secondary antibodies (2.1.15) diluted in blocking solution for 45 min at
room temperature in a moist chamber. For the staining of cell nuclei, slides were incubated
in DAPI solution diluted 1:1500 in PBS for 10 min at room temperature in a dark chamber
and again washed twice with PBS. Following that, slices were mounted with Aqua
Polymount (Polysciences Inc. Warrington, USA).
2.2.8.9 Detection of -galactosidase in tissue sections (X-Gal staining)β
The bacterial enzyme β-galactosidase catalyzes the cleavage of the O1 bond of the
sugar β-D-galactose to a substituent. Due to broad substrate specificity the enzyme can also
be used to cleave organic compounds such as 5-bromo4-chloro-3-indolyl-β-D-galactoside
(X-Gal) giving rise to an indigo-colored precipitate (5-bromo-4-chloro-3-hydroxyindole)
under oxidizing buffer conditions. Furthermore, this enzyme can be expressed in
mammalian cells when placed under the appropriate regulatory elements and is usually
well tolerated. This property of the enzyme allows the use of β-galactosidase in transgenic
mice as a reporter for the detection of transgene activity or for the detection of promoter
activity of an endogenous gene in ‘knock-in’ approaches (Goring et al, 1987; Sanes, 1994).
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MATERIALS AND METHODS
To detect -galactosidase activity in transgenic mice, slides containing 14- mβ μ
vibratome sections were washed in LacZ washing solution (2.1.11) for 10 min at room
temperature. Slides were then washed in PBS thrice for 15 min each. Next, the slides were
placed in LacZ staining buffer (2.1.11) for 20 min to 24 hours at 37 °C in the dark. The
following day, sections were washed in PBS thrice for 15 min each which stopped the X-gal
reaction. The slides were mounted with Aqua Polymount. Tissue sections can be stored for
more than 1 year at room temperature in the dark without any detectable loss of tissue
integrity or diffusion of the indigo-colored precipitates.
2.2.8.10 Confocal microscopy, image acquisition and analysis
Cells were analyzed using Axioimager (Leica Microsystems, Bensheim, Germany).
Pictures were obtained at 1024x1024 pixel resolution using 20x, 40x and 63x magnification
objectives. The images were analyzed with Leica TCS-NT software. Merges were also
obtained using the same software. Images were further developed and organized by Adobe
Photoshop and Illustrator software (Adobe, San Jose, USA). Image quantification was
performed using ImageJ software (NIH, USA).
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MATERIALS AND METHODS
2.2.9 Embryonic stem cell (ES) culture
2.2.9.1 Culture of Embryonic stem cells
ES cells were routinely grown on a monolayer of MEF cells at 37 °C in a 5 % CO 2
atmosphere. Only low passages of ES cells were used (less than 15 passages), since longer
culture times adversely affect ES cell pluripotency. Cells were kept at a relatively high
density and the medium was changed every 24 hr. Cells were passaged when colonies
reached ∼60 % confluency. The plate was washed twice with prewarmed ES-HBSS,
incubated with ES trypsin/EDTA for 2 min at 37 °C in a 5 % CO2 atmosphere. The cell
aggregates were disrupted by pipetting with a P200 Gilson pipette five times and
subsequent incubation for 1 min at 37 °C. Trypsinization was stopped by adding ES culture
medium (2.1.9). The cell suspension was transferred to a 15 ml falcon tube and ES medium
was added to a final volume of 10 ml. The cells were pelleted for 5 min at 800 x g at room
temperature, resuspended in ES medium (with or without geneticin depending upon the ES
cells), and plated on a fresh MEF monolayer.
2.2.9.2 Thawing of ES cells
The vial containing the frozen ES cells was allowed to thaw rapidly in a 37 °C water
bath. When the cells were nearly thawed, they were transferred into a falcon tube
containing 10 ml of prewarmed ES cell culture medium. The cells were pipetted up and
down to break apart cell clumps. The cells were then centrifuged at 800 rpm, 22 °C for 5
min. The medium was carefully sucked off from the sides of the falcon and the pellet was
redissolved in 1 ml of prewarmed ES culture medium. The cells were then carefully seeded
on 6-well feeder-coated plates. The cells were allowed to settle down for 5 min at room
temperature and then carefully transferred to a cell culture incubator at 37 °C with an
atmosphere of 5 % CO2.
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MATERIALS AND METHODS
2.2.9.3 Preparation of mouse embryonic fibroblasts (MEF’s)
Pregnant CD1-MTK neo female mice were sacrificed at E13-E14 by cervical
dislocation after anaesthetization. The embryos were removed from the uterus, and after
cutting away the brain and dark red organs, were washed with fresh PBS in order to
remove as much blood as possible. The remaining tissue was minced with two scalpels,
cells/tissue were suspended in several ml of prewarmed trypsin/EDTA (about 1-2 ml per
embryo) and incubated with gentle shaking at 37 °C for 20 min. The suspension was
transferred to a falcon tube containing a suitable volume of prewarmed DMEM/10 % (v/v)
FCS and filtered through a nylon mesh (pore size 50 µm). The filtrate was subjected to low
speed centrifugation (800 rpm, 5min). The resulting cell pellet was resuspended in
prewarmed MEF medium and plated out at 1 embryo equivalent per 1 T75cm flask. The
flasks were precoated with 0.5 % gelatin before seeding MEF cells. The medium was
changed on the following day, with the fibroblasts being the only cells that attached to the
dishes. The fibroblast cultures were passaged for maximum 5 times before irradiation.
2.2.9.4 Feeder irradiation
Plates containing MEF cells were confluent within one to a few days after plating.
When the MEFs formed a confluent monolayer, they were trypsinized and replated in a T-
75 cm2 flask. When the flask was confluent, cells were trypsinized and either frozen in MEF
freezing medium or replated in 3 x T-75 cm2 flasks. After the cells had reached confluency,
the flasks were washed with HBSS, trypsinized, and splitted to a T-175 cm2 flask/each T-75
cm2 flask. Upon cell confluence, the feeders were trypsinized and pooled in 50 ml of MEF-
medium and treated with 3000 rads of gamma irradiation to inhibit cell growth and
division. The inactivated MEFs were then either frozen in MEF freezing medium (3 x 1 ml
aliquots per confluent T-75 cm2 flask) and stored at -80 °C or used for plating cell culture
plates for the culture of ES cells. For using the irradiated feeders, 1 vial of the feeders was
plated on a 0.5 % gelatin-precoated 1 x 6-well plate.
2.2.9.5 Freezing of cells
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MATERIALS AND METHODS
After trypsinization, ES cells or MEFs were pelleted for 5 min at 800 x g at room
temperature and resuspended in the appropriate volume of prechilled freezing medium (3
ml of MEF freezing medium for one confluent T-125 cm2 flask; 1 ml of ES freezing medium
for one confluent well of a 6-well plate). The cell suspension was transferred to prechilled
1-ml cryovials containing 500 µl of the respective freezing medium (2.1.9) and rapidly
frozen on dry ice or transferred quickly to a pre-cooled freezing device at -80 °C. The cells
were stored for 2 days to 2 weeks at –80 °C. For long-term storage, the cells were
transferred to liquid nitrogen tanks.
2.2.9.6 Electroporation of the transgene construct into ES cells
Plasmid DNA of the transgene vector was prepared according to the Plasmid maxi kit
manual and the DNA was linearized by restriction enzymes SfiI and Eam11051 to remove
the excess vector backbone. After digestion, the vector DNA was precipitated, washed twice
with sterile 75 % (v/v) ethanol, and then dissolved in electroporation buffer (2.1.10). All
steps were performed under sterile conditions using sterile consumables under the hood.
ES cells from 4 confluent wells of a 6-well culture plate were trypsinized with 0.25 %
trypsin/EDTA for 5 min at 37 °C. The reaction was stopped by adding 3 ml of prewarmed ES
cell medium. The cells were pipetted up and down to break apart any cell clumps. The cells
were then pelleted for 5 min at 800 x g at room temperature. The cell pellet was
resuspended in 500 µl of prewarmed sterile electroporation medium and transferred into a
0.4 cm electroporation cuvette (Biorad). The final volume of the suspension was made up to
1 ml including the amount of DNA to be added for the electroporation. 20 µg of linearized
DNA were added to the cell suspension and incubated for 5 min at room temperature. The
cuvette was then placed in the electroporation chamber (Biorad Gene Pulser) and
electroporated using the following settings: 500 µF, 240 V, = 5.7-6.0 ms. The cells wereτ
allowed to recover for 10 min at room temperature, transferred to 100 ml of prewarmed ES
culture medium, mixed and distributed equally into 10 cm dishes with feeder cells and
incubated at 37 °C with an atmosphere of 5 % CO2. After 24 hr, the ES medium was replaced
with ES cell selection medium containing geneticin. The ES cells were allowed to grow in
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MATERIALS AND METHODS
the selection medium for 5-7 days. The medium was exchanged regularly to allow proper
growth of the cells.
2.2.9.7 Selection of ES cell clones
One day before isolating ES cell colonies, 24-well plates with feeder cells were
prepared. The plates with electroporated ES cells were washed twice with 8 ml of
prewarmed HBSS. The cells were left in the second HBSS wash during picking. A P-200
pipette with 1-200 µl filter tips was used to pick individual colonies. First, the drug-
resistant ES cell colonies were slightly dislodged with the pipette tip containing 20 µl HBSS
and then transferred to a well of a 96-well plate. After picking 24 clones, 80 µl of
trypsin/EDTA was added to each well and the cells were incubated at 37 °C for 1 min. 10-20
pipetting cycles were used to generate single cell suspension by using a multichannel
pipettor. Then 80 µl ES cell selection medium was added to each well and mixed. The whole
volume of the solution was then transferred into 24-well feeder plates. The same procedure
was repeated until all clones in good shape had been isolated. Around 480 ES cell clones
were isolated and cultured.
2.2.9.8 Preparation of DNA from ES cells
500 µl of ES cell lysis buffer (2.1.9) was added to each well containing growing ES
cells in 24-well plate and incubated at 37 °C for 2-4 days. The lysate was transferred to a
microcentrifuge tube and 1 volume of isopropanol was added to precipitate the genomic
DNA. The DNA was then picked up with a pipette tip and transferred to a clean
microcentrifuge tube. The precipitated DNA was washed with 100 µl of 70 % EtOH. Tubes
were centrifuged at 1000 x g at room temperature and the precipitated DNA was dissolved
in 50 µl of low TE.
2.2.9.9 DAB staining of the ES cells
To check for the expression of EGFP and mycGlyT1 upon cre recombinase
expression, DAB staining was performed on ES cell clones. Selected ES cell clones were
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MATERIALS AND METHODS
grown on coverslips in 24 well plates till they reached confluence. To activate the transgene
in vivo, cells were electroporated with 15 µg of pGKCre plasmid and allowed to express for 2
days. The cells were fixed in 4 % PFA solution and treated with 0.3 % H2O2 in PBS for 30 min
to limit endogenous peroxidase activity. After washes with PBS, cells were blocked in
blocking solution (2 % BSA, 1 % NGS, 0.3 % Triton X) for 1 hr. For staining, cells were
incubated with rabbit anti-EGFP antibody for 1 hr. After 3 washes with PBS, cells were
incubated with anti-rabbit biotinalated secondary antibody for 1 hr. For DAB staining, cells
were incubated in a mixture of solution A and solution B (Vecstatin ABC kit, Vector
Laboratories, Burlingame, USA) for 2 hr at room temperature. For the DAB reaction, 1 tablet
of DAB (Sigma) was dissolved in 70 ml of 0.05 M Tris-Cl, pH 7.5 and filtered through a 0.45
µn filter. 3.5 µl of H2O2 was added to this solution and the coverslips were incubated in the
solution till the brown precipitate developed. The reaction was stopped by immersing the
coverslips in a solution of 0.05 M Tris-Cl, pH 7.5. For mounting the coverslips, they were
desiccated through a series of 70 %, 90 %, 100 % ethanol, and Xylene. The coverslips were
then mounted using . Respective non-cre electroporated ES cell clones were taken as
negative control for this experiment.
2.2.9.10 Preparation of ES cell clones for blastocyst injection
A week prior to injection, the frozen ES cells were thawed in 24-well plates with
feeder cells and expanded from 24-well to 6-well plates. The ES cells with small compact
colonies that were in logarithmic growth phase were selected for injection. On the day of
injection, the cell medium was changed and the cells were washed twice with prewarmed
HBSS. The cells were trypsinized by adding trypsin/EDTA. The cells were incubated at 37 °C
for 3-8 min to allow the dissociation of the cells from the plate. Trypsinization was stopped
by adding 1-2 ml of prewarmed ES cell selection medium and ES cell aggregates were
disrupted by pipetting 10 times. 7 ml of the prewarmed ES cell medium was added to make
a cell suspension. The cells were then centrifuged for 5 min at 1000 rpm and room
temperature. The supernatant was discarded and the cells were resuspended in 1 ml of
prewarmed ES cell medium. The suspension was then plated on gelatinized 6 cm plate
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MATERIALS AND METHODS
containing 4 ml of the ES cell medium. The cells were incubated at 37 °C for 45 min to allow
the feeders to sediment. The plate was then washed briefly with the supernatant and ES
cells plated on a fresh gelatinized 6 well plate. The cells were again incubated at 37 °C for
30 min. The plate was visualized under a microscope and if there were too much feeders
left, the above protocol was repeated for the third time. Finally, the plate was washed
briefly with the supernatant and the cell suspension centrifuged for 5 min at 1000 rpm and
room temperature. The cells were then resuspended in 1 ml of injection medium (ES
medium + LIF + 20 mM HEPES). The injection of the ES cells into the blastocyst was
performed by Frank Zimmerman at University of Heidelberg, Germany.
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MATERIALS AND METHODS
2.2.10 Glycine uptake experiments
2.2.10.1 Brain membrane preparation
Mouse brain samples were homogenized in 1 ml of ice-cold P2 prep membrane
preparation medium (2.1.11) using a Dounce-type glass/teflon homogenizer. The
homogenate was centrifuged (1000 x g, 5 min) at 4 °C. The pellet (P1) was discarded, and
the supernatant was centrifuged (17000 x g, 10 min) at 4 °C. The resulting pellet (P2) was
washed once with ice cold modified Krebs-Henseleit medium (2.1.11). The mixture was
centrifuged (17000 x g, 5 min) at 4 °C and the final resulting pellet was resuspended in ice
cold modified Krebs-Henseleit medium. Protein concentrations were determined using
Bradford protein assay system (Bio-Rad).
2.2.10.2 (3H) Glycine Transport Assay
20 µl aliquots of the membrane suspension (equivalent to 30–50 µg of protein) in 80
µl of Krebs-Henseleit medium were preincubated for 2 min at 37 °C. All experiments were
performed in triplicates Uptake was initiated by addition of 100 µl of modified Krebs-
Henseleit solution kept at 37 °C containing [3H]-glycine (2 µM) (1.52 GBq/µmol,
MovasekInstru Biochemicals, Brea, CA). After 1 min incubation with gentle agitation, uptake
was terminated by diluting the incubation mixture with 3 ml of ice-cold modified Krebs-
Henseleit medium followed by rapid filtration through a moistened filter (SM 111060, 45
µm pore size, Sartorius, Gottingen, Germany). Filters were rinsed twice with 3 ml of
modified Krebs-Henseleit medium. All dilution, filtration, and washing procedures were
performed within 15 s. Filters were dried and placed in microvials, and their radioactivity
measured by scintillation counting (Beckmann, Germany). The glycine mediated uptake
was calculated as follows
Relative uptake = cpm/ [total glycine concentration] (pmol) Total protein conc. (mg) * time (min)
2.2.11 Protein biochemistry methods
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MATERIALS AND METHODS
2.2.11.1 Protein determination by Bradford assay
The protein concentration of the samples was measured using the Bio-Rad ‘Bradford
Protein Assay’ The Bradford assay, a colorimetric protein assay, is based on an absorbance
shift of the dye Coomassie Brilliant Blue G-250 under acid conditions when a redder form of
the dye is converted into a bluer form on binding to protein. The binding of the protein
stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in
solution is a measure for the protein concentration, and can be estimated by use of an
absorbance reading. The (bound) form of the dye has an absorption spectrum maximum
historically held to be at 595 nm. The increase of absorbance at 595 nm is proportional to
the amount of bound dye, and thus to the amount (concentration) of protein present in the
sample.
Due to presence of high lipid content in the membrane preparations, the protein
determination assay was modified. In short, in a microcentrifuge tube, 1 µl of the protein
sample was mixed with 49 µl of 0.2 N NaOH. 750 µl of H2O and 200 µl of the Bio-Rad reagent
were added to the mixture. The mixture was vortexed thoroughly. Subsequently the
absorption was read at the wavelength of 595 nm. The protein concentration could be
determined by reference to a curve. We used different dilutions of bovine serum albumin
(BSA) in 0.2 N NaOH as a reference control to plot the reference curve.
2.2.11.2 Discontinuous SDS Polyacrylamide Gel Electrophoresis
The most widely used denaturing and discontinuous polyacrylamide gel
electrophoresis (SDS-PAGE) method for protein separation was described by Laemmli
(Laemmli, 1970). In this method buffers of distinctive pH and composition generate a
discontinuous pH and voltage gradient in the gel. The discontinuity in pH and voltage
concentrates proteins in each sample into narrow bands thereby allowing the separation of
very dilute samples. The protocol primarily relies on denaturing proteins by heating in the
presence of SDS and β-mercaptoethanol (β-ME). Under these conditions, the subunits of
proteins are dissociated and their biological activities are lost. Most proteins bind SDS in a
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MATERIALS AND METHODS
constant SDS-to-weight ratio, leading to identical charge densities for the denatured
proteins. Thus, the SDS-protein complexes migrate in the polyacrylamide gel according to
size, not charge. Most proteins are resolved on polyacrylamide gels containing from 5 % to
15 % acrylamide and 0.2 % to 0.5 % bisacrylamide. The detailed theory and protocol for
one dimensional gel electrophoresis has been described in Gallagher, 2006; Hames, 1990.
A glass plate and a 1mm spacer plate sandwich of the electrophoresis apparatus was
assembled according to Bio-Rad instructions. The separating gel solution of desired
percentage of acrylamide (8%-12%) was prepared freshly and poured along an edge of one
of the spacers until the height of the solution between the glass plates was 2/3rd of the
maximum height of the glass plates. The top of the gel was slowly covered with a layer (1
cm thick) of isopropanol. The gel was allowed to polymerize for 30 min at room
temperature. Once the gel had polymerized, the layer of isopropanol was poured off and
was twice rinsed with ddH2O to remove any residual isopropanol. The stacking gel solution
was freshly prepared and was poured slowly on top of the polymerized separating gel along
an edge of one of the spacers until the solution reached the top of the plates. A 1 mm Teflon
comb (10 teeth) was inserted into the layer of stacking gel solution. The stacking gel
solution was allowed to polymerize for 30 to 45 min at room temperature. After
polymerization, the teflon comb was removed carefully. After the comb was removed, wells
were rinsed with 1X SDS electrophoresis buffer.
For loading of the samples, 1/4 volume of SDS loading buffer (4X) was added to the protein
samples and incubated at room temperature (for analysis of membrane transporter
proteins) for 15 min with shaking. The samples were then centrifuged at 13000 rpm for 15
min. The centrifuged samples were then directly used for analysis. For analysis of soluble
proteins, the samples were heated at 95 °C for 5 min. 20-40 µg of the protein sample was
loaded per well. Electrophoresis was carried out at 25 mA per gel for 40 mins. 5 µl of See
Blue Plus2 marker (2.1.5) was used as molecular weight standard. After the loading buffer
had eluted out of the separating gel the power supply was disconnected. The gel was
carefully removed and subjected to protein blotting.
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MATERIALS AND METHODS
2.2.11.3 Western blot analysis
Western blot is an extremely useful tool for the identification and quantification of
proteins in complex mixtures of proteins that are not radiolabeled. In this technique,
electrophoretically separated protein samples (2.2.11.2) are transferred from a gel to a
membrane and probed with antibodies that react specifically with a particular antigenic
epitope. Proteins can be detected down to femtomole quantities, well below the detection
limit for most assay systems.
Following electrophoresis, the separated proteins were transferred from the SDS
polyacrylamide gel to the membrane (2.1.6) by electrotransfer using a Mini-Trans-Blot
electrophoretic chamber (Bio-Rad; Munich, Germany). The transfer was carried out in
transfer buffer (2.1.12) at 30 V O/N or at 100 V for 2 hr on ice. After the transfer, the
membrane was stained for 5 min in Ponceau solution to visualize the transferred protein
bands.
2.2.11.4 Immunodetection of blotted proteins
The specific identification of protein bands was performed by indirect
immunodetection. To avoid unspecific binding of the primary antibody, the membrane was
incubated in blocking solution (2.1.12) for 1-2 hr at room temperature with shaking. The
membrane was then incubated, either overnight at 4 °C or for 1 hr at 25 °C in blocking
solution containing the desired dilution of the primary antibody (2.1.15). After 3 washes
with 1X PBS/0.05 % (v/v) Tween-20 for 10 min each, the membrane was incubated with
the secondary antibody conjugated to HRP diluted in blocking buffer for 45 min at room
temperature. The blots were again washed thrice with 1X PBS/0.05 % (v/v) Tween-20 and
HRP coupled secondary antibody was directly detected using the SuperSignal West Pico
Chemiluminescent Substrate kit (Pierce, USA) following the manufacturer’s instructions.
HRP is an enzyme that catalyzes the oxidation of luminol-based substrate leading to an
excitation of the chemiluminescent substrate that generates light at the site of reaction,
which is visualized through exposure to an X-ray film. The membrane was then covered
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MATERIALS AND METHODS
with a transparent film and an ECL photographic film (Hyperfilm™, Amersham Biosciences)
was exposed to the membrane. The time of exposure varied from 1 sec to 15 min depending
on the signal intensity. Afterwards, films were developed by a Kodak X- OMAT 2000
processor (Kodak, Atlanta, USA).
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3 RESULTS (PART I)
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3.1 Role of Glycine and Glycine transporters in the CNS
Glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem,
acting at strychnine-sensitive GlyR’s-chloride (Cl-) channels (Legendre, 2001). At these
glycinergic inhibitory synapses, the post-synaptic actions of glycine are terminated by a
rapid reuptake mechanism, which is mainly mediated by glycine transporters (GlyT’s).
GlyT’s which include GlyT1 and GlyT2, belong to the family of sodium/chloride dependent
transporters (Eulenburg et.al., 2005). GlyT1 is widely expressed in astrocytic glial cells
whereas GlyT2 is localized to the presynaptic terminals of glycinergic neurons
predominantly in brain stem and spinal cord (Zafra et.al., 1995 a,b).
Apart from acting as a classical inhibitory neurotransmitter, glycine is unique in that it also
acts along with glutamate, as an essential co-agonist of the excitatory NMDA receptors
(NMDAR’s). In higher regions of the brain such as hippocampus, glycine has been shown to
simultaneously increase Gly-R mediated inhibition and facilitate NMDAR-dependent
plasticity at excitatory synapses (Zhang et.al., 2008). The simultaneous activation of
excitatory NMDAR’s and inhibitory GlyR’s may provide a homeostatic regulation of
hippocampal network function.
In the hippocampus, GlyT1 is mainly expressed by astrocytes and neuronal GlyT1 is only
found at glutamatergic synapses (Cubelos et.al., 2005). At these synapses, it is postulated
that astrocytes via GlyT1 are the major source of hippocampal glycine (Yang et.al., 2003).
However, a direct evidence for the release of glycine via this transporter in still lacking. To
understand more about the regulation of glycine (release/or uptake) in more frontal
regions of the brain, I generated an inducible GlyT1 transgenic mouse line which is
described in this thesis.
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3.1.1 Considerations for construct design for generation of an inducible
(in)GlyT1 transgenic mice
By “classical” definition, a transgenic organism carries extra, often foreign (i.e. from a
different organism) DNA into its genome called a transgene. This transgene can be
integrated into the host genome either randomly, via viral injection or by creating a site-
specific ‘knockin’.
In “classical transgenesis,” the DNA of interest is sub-cloned downstream of a suitable
promoter element that drives its expression, followed by a stop signal to stop transcription.
To a large extent, this promoter element determines the level, tissue specificity, and
temporal pattern of transgene expression. For e.g. for exerting control over the expression
of the transgene in a cell-type or tissue specific manner, tissue or cell-type specific
promoters are used, (Glial Fibrillary Acidic Protein (GFAP) promoter, specific for glial cells).
In contrast, for a more ubiquitous expression, ubiquitin promoter and -actin promoter areβ
more commonly used. Reporter genes encoding proteins, such as GFP, LacZ are cloned
downstream of the promoter as a read out for the promoter activity in a cell or a developing
animal. (Voncken and Hofker, 2005). Regulatory elements such as viral enhancers are also
commonly used in the transgenic constructs to enhance transgene expression and achieve
position-independent and copy number dependent expression of the transgene.
The problem encountered during “classical transgenesis” is that most of the animals show
high variation in expression pattern due to random insertion of the transgene in the
genome, including undesirable expression of the transgene in a particular tissue(s) (ectopic
expression). This could lead to phenotypes that nonspecifically affect the nature of the
system in question and can lead to results which might not be due to the transgene
influence. Another problem arises due to sensitiveness of the expression of the transgene to
its proximity to transcriptional activators and silencers. Silencer elements are distributed
throughout the genome, making it possible that some integration sites yield little or no
expression of the transgene.
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To overcome the problems posed by classical transgensis, temporal control over transgene
expression is required. This is achieved by an inducible system wherein the expression of
the transgene can be turned on or off at defined times and positions.
“Conditional transgenesis” is used to have a better control over the (trans) gene expression
in a given organism and is integrated by the use of tissue-specific promoters and/or control
element usage (Voncken and Hofker, 2005). Due to this, transgene expression is confined to
a defined target tissue or selected cells within a tissue. Conditional transgenesis employs
the use of “binary switches” that can be used to turn “on” or “off” the transgene. Two types
of binary switch models are commonly used to conditionally induce the transgene.
a) Tetracycline controlled transgene expression: the Tet-system: this system is based
upon the tetracycline resistance operon from E. coli. Inducibility in this system
depends on the presence or the absence of tetracycline or its analog doxycycline. In
the “tet-off” system, presence of tet or doxycycline prevents the expression of the
transgene by effectively binding to the tTA. This is in contrast to the “tet-on” system
where the presence of tetracycline allows the expression of the transgene. Despite
its convenience for inducing transgene expression, this system suffers from some
disadvantages. Firstly, the requirement to combine two transgenic lines often leads
to unexpected patterns of transgene expression. In addition to regional variability, a
line may also show chimeric expression within a given region (Yamamoto et.al.,
2001). Another disadvantage of the system arises from the exposure of the animals
to tetracycline. To induce gene only in the adult animal requires the persistent
exposure (beginning at the conception) of the mice to tetracycline to prevent
transgene expression. This can be expensive and the effects of the long term
exposure to tetracycline are unclear. Also, conditional control of transgene
expression in Furthermore, tetracycline clears slowly away from the animal thereby
delaying transgene expression after long term exposure (Yamamoto et.al., 2001).
This makes this system unsuitable for the investigation of acute transgene effects.
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b) Transgene induction by the removal of a transcriptional block: Cre/loxP system: This
system makes use of a recombination process derived from bacteriophage P1 as
explained in (cross-reference to the introduction). Here an on/off transgenic system
employs a silenced transgene, in which a strong transcriptional block is bracketed by
two similarly oriented loxP sites. The DNA sequence flanked by loxP sites is said to
be “floxed.” A second independent transgenic line expresses cre recombinase under
a tissue specific promoter. Cre recombinase-mediated recombination results in the
removal of entire floxed element, leaving one loxP site behind and thereby deleting
(knockout) or expressing (knockin) the region of interest. However, the expression
of the transgene is unidirectional i.e. once the expression is put “on” it can’t be put
“off.”
One obvious advantage of the Cre/loxP system lies in the fact that it can be used to generate
mice lacking a protein in a particular tissue to avoid early lethality or severe developmental
consequences. Furthermore, this system allows for a spatial and temporal control over
transgene expression and takes advantage of the inducers with minimal pleiotropic effects.
For these reasons we employed the use of this system for the generation of the inGlyT1
transgenic mice which would allow for the time and cell/ tissue specific activation of the
transgene. For the generation of the transgene construct, a ubiquitous promoter along with
a strong enhancer element was chosen which could constitutively and ubiquitously express
higher levels of the transgene in all cell types. A floxed reporter silencing cassette was
cloned downstream of the promoter to allow for the selection of the cells harboring the
construct. cDNA of the gene of interest was cloned downstream of the floxed cassette. A
second reporter was inserted downstream of the cDNA of interest to act as a marker for the
removal of the floxed cassette and the expression of the cDNA.
The inducible expression of the transgene construct works in the following way: the
introduction of the transgene into the host cells leads to the expression of the reporter in all
cell types under the ubiquitous promoter. This is being referred to as “silenced construct.”
Expression of cre recombinase under a cell or tissue specific promoter leads to the excision
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of the floxed cassette and the expression of the transgene. This is referred to as “cre-
recombined construct.” Since GlyT1 is expressed upon cre excision, it is also referred to as
inGlyT1. A second reporter is used to detect the deletion of floxed cassette and the
expression of cDNA of interest. The Internal Ribosome Entry Site (IRES) element present in
the vector allows the bicistronic expression of the gene of interest and the second reporter
separately. IRES drive the translation of the cDNA of interest and reporter independently
such that both the proteins are expressed in the same cell. (Fig. 3.1).
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Fig. 3.1: Strategy for the generation of inGlyT1 transgenic miceThe expression of GlyT1 in the transgene construct is inducible. The silenced construct expresses LacZ as a first marker prior to cre excision. Expression of GlyT1 is prevented by a poly-A signal downstream of LacZ/Neor cassette. The expression of GlyT1 is accomplished by the expression of cre recombinase which recognizes the two loxP sites in the construct and thus removes the silencing cassette. This is known as “cre-recombined construct.” EGFP is expressed as a second reporter marker after cre excision.
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3.1.2 Generation of the transgene construct
The expression vector used for the generation of the inGlyT1 mouse line was based
on an approach from Lobe et.al., 1999 and
Please_Select_Citation_From_Mendeley_DesktopNovak et.al., 2000 The expression of the
transgene is driven by ubiquitously active chicken -actin promoter. An enhancer elementβ
cassette (CAG cassette) from Cytomegalovirus (CMV) (Xhu et.al., 2001, Niwa et.al., 1991) is
cloned upstream of the promoter to direct strong expression. Expression of the transgene
GlyT1 is prevented by the expression of a silencing cassette containing a first reporter, a
LacZ/Neor fusion protein; (Friedrich and Soriano, 1991) that was followed by a triple
repeat of the simian virus (SV40) polyadenylation signal. The reporter along with the stop
signal is flanked by two loxP sites. This allows the removal of the silencing cassette upon
expression of cre recombinase (Fig. 3.2, B). The original vector map is listed in appendix III.
Transgene construct was generated by cloning the GlyT1 cDNA downstream of the floxed
LacZ/Neor cassette via Bgl II and Xho I restriction sites in the vector (Fig. 3.2, A). An IRES
was inserted downstream of the GlyT1 cDNA to allow for bicistronic expression of EGFP
(Enhanced Green Fluorescent Protein) derived from the jellyfish, Aequorea victoria (Chalfie
et.al., 1994) and GlyT1. This construct was designated as iLacZ/GlyT1-EGFP.
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Fig.3.2: Schematic drawing of iLacZ/GlyT1-EGFP construct used for generating transgenic mice (A). GlyT1 cDNA was cloned using Bgl II/Xho I restriction sites into the pCCALL2-IRES-EGFP/anton vector. (B) Transgene expression is driven by the ubiquitously active CMV-enhanced chicken ß-actin promoter. A floxed LacZ/Neor cassette serves as a selection marker and a silencing cassette due to a poly-A signal at its 3' end. Following that is coding sequence for GlyT1 and EGFP. LacZ reporter is used as first marker, expressed prior to Cre excision, and the EGFP reporter as the second marker, expressed after Cre excision.
For the ease of understanding the terms used in this study, the “silenced construct” will be
referred to as iLacZ/GlyT1-EGFP and the “cre-recombined” construct as i LacZ/GlyT1-Δ
EGFP Characterization of tagged GlyT1 constructs
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In order to distinguish between the endogenously and transgenically expressed
GlyT1, a DNA sequence encoding for C-myc epitope was introduced into N-terminus (GlyT1-
N) and C-terminus (GlyT1-C) of the GlyT1 cDNA respectively (Fig. 3.3, A). The tagged GlyT’s
(mycGlyT1) were cloned into vector pcDNA3.1 and their functional properties were
analyzed by glycine uptake experiments and Western blotting (Fig. 3.3, B, and C) in HEK
293 cells.
Glycine uptake experiments were performed after 2 days as described in section 2.2.10.2.
The glycine uptake experiments revealed no significant difference between the uptake
activities of cells transfected with tagged and untagged GlyT1 constructs. Both tagged
constructs showed a concentration-dependent uptake of radiolabeled glycine, although
GlyT1-N showed much less uptake activity than GlyT1-C. Untransfected HEK 293 cells and
cells transfected only with pcDNA3.1 also showed minimal glycine uptake activity which
was much less than cells expressing the transporter constructs (Fig.3.3, B). Thus, it can be
concluded that the tagged transporter constructs were functional and can transport glycine.
For western blot analysis, lysates from HEK 293 cells transfected with different constructs
were prepared as described in section 2.2.8.5 and detected using anti-myc and anti-GlyT1
antibodies (Fig. 3.3, C). The cells expressing both myc-tagged constructs were recognized by
anti-myc and anti-GlyT1 antibodies respectively. When probed with anti-myc antibody,
immunoreactivity was detected for GlyT1-N as well as GlyT1-C whereas the untransfected
and cells expressing wildtype GlyT1 didn’t show any immunreactive bands. On probing
with anti-GlyT1 antibody, cells expressing both myc tagged constructs and endogenous
GlyT1 were recognized. The different band sizes at 110 kDa, 98 kDa and 58 kDa depict the
different complex glycosylated and unglycosylated forms of GlyT1 (Fig. 3.3, C). Since N-
terminally tagged construct showed glycine dependent uptake activity and was more
reliably detected in the western blots, it was further chosen for the generation of inGlyT1
transgenic mice.
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Fig 3.3: Characterization of the tagged GlyT1 constructs(A) Depict the membrane topology of glycine transporter 1 with tags inserted at N and C terminus of the glycine transporter 1 (purple and red circles) respectively. (B) Depict 3 [H] glycine uptake activity by different constructs when expressed in HEK 293 cells. (C) Western blots analysis of membrane lysates expressing differently tagged constructs when probed with anti-myc and anti GlyT1 antibodies. (Original uptake and western blot data from Chigusa Shimizu Okabe).
3.1.3 Verification of the transgene construct
After the generation of the transgenic construct, different regions of it were verified
using PCR, restriction digest analysis and sequencing.
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PCR was performed using primer pair 1S/2AS to check for the correct insertion of
mycGlyT1. The binding sites for the primers are listed in appendix I. The primer pair
resulted in a fragment of 727 bp from vector iLacZ/GlyT1-EGFP in which GlyT1 was cloned.
The corresponding original vector, pCCALL2-IRES-EGFP/anton which lacked GlyT1, didn’t
show any amplified product (Fig. 3.4, A). The amplified fragment was cut from the gel and
sequenced to check for the correct sequence of the fragment. The sequenced fragment was
verified by aligning it to the original GlyT1 cDNA sequence using Sequencher (Ann Arbor,
U.S.A) (data not shown). The sequencing confirmed the correct insertion of mycGlyT1 into
iLacZ/GlyT1-EGFP vector.
Different enzymes were chosen for restriction digest analysis upon prediction analysis by
the program MacVector (Cambridge, UK). Single as well as multiple base cutters were
chosen to digest the plasmid. The complete restriction map of the vector as well as
predicted fragment sizes is given in appendix IV. Single base cutter SfiI, linearized the
plasmid. (Fig.3.4, B). A double digest with SfiI/AhdI and SfiI/Eam11051 released a fragment
of 1273 bp. Multiple cutters ∼ ApaI, ClaI and SpeI also released expected size fragments.
However, digests with PstI and NotI were spurious and unpredicted fragment sizes were
obtained (Fig. 3.4, B). Restriction analysis with other enzymes gave fragments as predicted
by the restriction map (data not shown).
To check for the presence of functional loxP sites in the construct, the plasmid was treated
with cre recombinase in vitro. In brief, the plasmid iLacZ/GlyT1-EGFP was treated with cre
recombinase (NEB) in 1X cre recombinase buffer at 37 °C for 30 min. The reaction was
stopped by incubating the mixture at 70 °C for 10 min. The plasmid was then transformed
into E.coli chemocompetent cells. DNA was isolated from the colonies obtained after
successful transformation.
If the two loxP sites present in the vector are correctly recognized by cre recombinase, then
it would lead to excision of the region between the loxP sites as depicted in Fig. 3.4, C. Two
single base cutters XhoI and XbaI were chosen to distinguish between the “silenced
plasmid” and “cre-recombined” plasmid. The restriction site for XbaI is located before the
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first loxP site and for XhoI after the second loxP site (Fig. 3.4, C). A “non-cre” recombined or
a “silenced construct” will release a fragment of 7538 bp upon an ∼ XhoI/XbaI digest.
However, if the loxP sites are recognized by the cre recombinase, the region between the
two loxP sites would be deleted and a digest with XhoI/XbaI will only release a fragment of
2567 bp.∼
As shown in Fig. 3.4, D, the restriction digest of the DNA isolated from transformed colonies
released a fragment of 2567 bp. Thus it can be concluded that the loxP sites present in∼
the iLacZ/GlyT1-EGFP vector were recognized by the purified cre recombinase which lead
to the recombination between the two sites, thereby excising the region in between them.
This implied for functional loxP sites in the vector which could be recombined in vitro.
Additionally, the transgene construct was sequenced using different primer pairs (as listed
in appendix I) and the acquired sequences were aligned using the software Sequencher
(Ann Arbor, U.S.A). The sequencing analysis showed a correct insertion of mycGlyT1 into
the vector. Different regions of the vector were also sequenced to check for any mutations
within the coding regions of different reporter regions and for the correct orientation of the
loxP sites (data not shown).
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Fig. 3.4: Validation of the transgene constructThe plasmid iLacZ/GlyT1-EGFP was verified via PCR and restriction digest analysis. (A) Show the PCR fragments obtained from the vector lacking inserted mycGlyT1 and one that contains mycGlyT1. (B) The restriction pattern obtained by the digestion of plasmid iLacZ/GlyT1-EGFP with different restriction enzymes. (C) Vector map showing the position of the loxP sites and cutting sites for XhoI and XbaI. (D) Verification of the functional loxP sites in the plasmid.
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3.1.4 Heterologous expression of the transgene construct in HEK 293 cells
After the transgene constructs was generated and verified, their functionality was
checked in HEK 293 cells. For the experiments, iLacZ/GlyT1-EGFP and I LacZ/GlyT1-EGFPΔ
plasmid DNA were transfected into HEK 293 cells (see 2.2.8.4) and the expression of the
markers, -gal and EGFP, were analyzed after 2 days of expression.β
To check for the cells expressing -gal encoaded by the LacZ gene in the construct, X-Galβ
staining of the transfected HEK cells was performed. X-Gal, also called as bromo-chloro-
indolyl-galactopyranoside, is an oragnic compound which acts as a substrate for the
enzyme -galactosidase. X-gal is cleaved by -galactosidase yielding galactose and 5-β β
bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5, 5'-dibromo-4, 4'-
dichloro-indigo, an insoluble blue product. This blue color can then be visualized by naked
eye. The EGFP fluorescence was analyzed by fluorescence microscopy after PFA fixation of
the cells.
The untransfected HEK 293 cells did not show any blue color upon treatment with X-Gal or
any immunofluorescence (Fig. 3.5, A1 and A2). In contrast, the cells expressing
iLacZ/GlyT1-EGFP showed blue color due to the expression of the enzyme -gal (Fig. 3.5,β
B1). In the same cells no EGFP fluorescence was detected since the expression of EGFP was
silenced (Fig. 3.5, B2). Cells transfected with I LacZ/GlyT1-EGFP Δ did not show any -galβ
expression due to the loss of LacZ/Neor cassette upon cre recombination (Fig. 3.5, C1).
However, these cells showed EGFP fluorescence where the soluble EGFP was accumulated
uniformly all over the cell when visualized by fluorescence microscopy (Fig. 3.5, C2). These
results showed that the reporters and the stop element were functional in the transgene
construct and the LacZ/Neor silencing cassette could be removed by cre recombinase in
vitro.
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Fig. 3.5: Functionality test of the constructs in HEK 293 cellsTo check for the functionality of the constructs, HEK cells were transfected with the indicated plasmids. Expression of -galβ or EGFP was analyzed by X-Gal assay or fluorescence microscopy. (A1, A2): untransfected HEK cells stained for -galβ and checked for EGFP fluorescence. (B1, B2): Cells transfected with iLacZ/GlyT1-EGFP show blue stained cells but no EGFP fluorescence. (C1, C2): HEK cells transfected with I LacZ/GlyT1-EGFP Δ , lacked -gal expression but show EGFP fluorescenceβ with the soluble EGFP accumulated all over the cell. Scale bar: 50µm
Immunostaining with different antibodies was also performed on fixed and permeabilized
transfected HEK 293 cells to check for the expression of the markers -gal, EGFP, andβ
mycGlyT1. Different dilutions of the antibodies which were used are listed in section 2.1.15.
To check for the expression of -gal, immunostaining with rabbit anti- -gal and Alexa 546β β
antibody was done. EGFP fluorescence was analyzed by fluorescence microscopy. The
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untransfected HEK 293 cells did not show any stained cells when probed with the antibody
(Fig. 3.6, A1, and C1). The cells transfected with plasmid iLacZ/GlyT1-EGFP showed
expression of -gal localized both in the cytoplasm as well as in the cell membrane (Fig. 3.6,β
A2, white arrows). These cells, however, did not express EGFP (Fig. 3.6, A2, and C2). This
can be explained by the fact that in plasmid iLacZ/GlyT1-EGFP, only -gal is expressed andβ
the expression of EGFP is silenced due to the presence of LacZ/Neor silencing cassette.
However, cells transfected with plasmid I LacZ/GlyT1-EGFP did not show any -galΔ β
immunofluorescence due to in vitro excision of the silencing cassette (Fig. 3.6, A3), but
express EGFP (Fig. 3.6, B3, and C3, white arrows).
Fig. 3.6: Immunostaining of HEK 293cells with anti -Gal antibodyβExpression of the markers -gal and EGFP upon staining with anti -gal antibody. (A1-C1) Showβ β untransfected HEK 293 cells; (A2-C2) and (A3-C3) depict staining of HEK 293 cells expressing iLacZ/GlyT1-EGFP and I LacZ/GlyT1-EGFP respectively. (B1-B3): depict the cell nuclei co-stainedΔ with DAPI. Scale bar: 50 µm.
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To analyze the expression of mycGlyT1 and EGFP, transfected HEK 293 cells were stained
with rabbit anti-myc antibody and anti-rabbit Alexa 546 (see 2.1.15). EGFP fluorescence
was analyzed by fluorescence microscopy as described previously.
In untransfected HEK 293 cells no myc immunreactivity was observed. Also, these cells did
not show any EGFP fluorescence or autofluorescence (Fig. 3.7, A1-D1). HEK 293 cells
transfected with plasmid iLacZ/GlyT1-EGFP also did not show any staining. This can be
explained by the fact that this plasmid contains the LacZ/Neor silencing cassette which
prevents the expression of mycGlyT1 and EGFP (Fig. 3.7, A2-D2). In contrast, the cells
transfected with plasmid I LacZ/GlyT1-EGFP show expression of mycGlyT1 as seen by theΔ
red channel (Fig 3.7, A3, white arrows). These cells also express EGFP (Fig 3.7, B3, white
arrows). Furthermore, it was observed that these two channels co-localize (Fig 3.7, C3,
white arrows), which meant that there was co-expression of both proteins in the same cell.
Together, these finding indicate that the stop element in the construct was functional since
there was no expression of mycGlyT1 and EGFP in cells transfected with iLacZ/GlyT1-EGFP.
Also, it proves that there is a bicistronic expression of both mycGlyT1 and EGFP since both
of these proteins can be detected together (Fig 3.7, D3, white arrows, yellow co-localized
dots) in the same cell transfected with I LacZ/GlyT1-EGFP.Δ
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Fig. 3.7: Detection of mycGlyT1 and EGFPFluorescence images showing HEK 293 cells transfected with iLacZ/GlyT1-EGFP and I LacZ/GlyT1-ΔEGFP plasmids respectively. Staining for the myc antibody is shown in red channel and for EGFP in green. Co-localization is shown by yellow color. (A2-D2) show cells transfected with iLacZ/GlyT1-EGFP and probed for myc and EGFP respectively. (A3) cells transfected with I LacZ/GlyT1-EGFPΔ show expression of mycGlyT1 and EGFP (B3). The expression of mycGlyT1 and EGFP is in the same cells as depicted by the yellow co-localization (D3). Scale bar 50µm.
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3.1.5 Backbone removal and linearization of construct
Before using the iLacZ/GlyT1-EGFP plasmid for the generation of the transgenic mice, a
part of its backbone was removed that was not necessary for the transgene functions. Two
restriction enzymes, SfiI and Eam1105 I were chosen which could delete the parts of the
plasmid which were not required. Restriction digest of the plasmid with these enzymes
removed parts of the vector backbone downstream of the second polyadenylation signal
(Fig. 3.8, A). Both these enzymes cut at single sites in the plasmid at positions 11225 bp and
12498 bp respectively
As depicted in the gel representative gel below, single restriction digest with both SfiI and
Eam1105 I linearized the plasmid iLacZ/GlyT1-EGFP (Fig. 3.8, B, lanes 2 and 3). However, a
double digest removes part of the vector backbone and releases a fragment of 1273 bp (Fig.
3.8, B, lane 4).
Fig. 3.8: Backbone removal and linearization of the plasmid iLacZ/GlyT1-EGFP(A) Shows the restriction map of the plasmid iLacZ/GlyT1-EGFP showing the location of restriction sites of the enzymes SfiI and Eam1105 I. (B) Representative gel showing the digestion of plasmid iLacZ/GlyT1-EGFP with SfiI and Eam1105 I.
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3.1.6 Genetic manipulation of the Embryonic stem (ES) cells
For the generation of the transgenic mice, the E14 (129/OLA) embryonic cell (ES)
line was used. The use of ES cells over the conventional technique of using transgene
constructs for pro-nucleus injections for the generation of the transgenic mice has several
advantages. For eg:
a) Single copy integrations of the transgene construct are more likely to be obtained by
electroporation in ES cells as compared to pro-nucleus injection where multiple
integrations of the transgene are more common. Single copy integrations are more
advantageous over multicopy integration since silencing of the transgene can occur
if cells have more than one copy of the transgene in their genome. If using a cre/loxP
system for the generation of the transgenic mice, then the recombination between
multiple loxP sites can pose problems by causing cryptic recombination events in
cells harboring the multicopy integrations and the desired recombination may not
be obtained.
b) ES cell clones can be tested before the generation of the transgenic mice for the
suitable expression of the transgene.
c) Only few mouse lines carrying the desired integration are obtained as compared to
pronucleus injection where high numbers of mouse lines have to be screened to
check for suitably expressing lines. If the transgene has been integrated into the
germ cells of the blastocyst embryos, then its highly probable that the expression of
the transgene would be maintained over generations (germ-line transmission).
The only limiting factors for generation of the mouse line with help of ES cells are that the
process is time consuming and for the mice must show a germline transmission to allow the
expression of the transgene to be analyzed over generations.
To maintain the ES cells in an undifferentiated state, they were cultured on the feeder layer
of irradiated mouse primary embryonic fibroblasts (MEF) cells in the presence of
recombinant leukemia inhibitory factor (LIF) in the medium.
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3.1.6.1 Isolation of ES cell clones
For the generation of the inGlyT1 mice, E14 ES cells (Fig. 3.9, A) were electoporated
with the linearized iLacZ/GlyT1-EGFP plasmid and grown for seven days under the
selection of G-418 (Fig. 3.9, B, and C). Since the LacZ/Neor fusion protein was expressed
under the control of the transgene promoter in the plasmid, G-418 resistant ES cell clones
were expected to express the transgene.
When the Neor ES colonies were large enough for expansion, those with undifferentiated
morphology were isolated and selected for further analysis. A total of 480 ES cell clones
were isolated out of which 400 survived which were then expanded. (Table 3.1).
Table 3.1: Summary of ES cell clones obtained
Description Number Percentage (%)
Total clones isolated 480
Surviving clones 400 83.33
Homogeneous clones 50 12.5
Non-homogeneous clones 250 62.5
Non-expressing clones 100 25
Once individual ES cell colonies were expanded, they were checked for the expression of the
transgene. X-gal staining of the expanded ES cells was performed to check for the
expression of the -gal. Some of the clones showed a homogeneous X-gal stain, while othersβ
were heterogeneous or didn’t express the transgene at all (Fig. 3.9, D). This could be
explained by the fact that the selection of the clones in the ES cell selection medium was not
complete. Since geneticin is utilized by the growing cells, they lower or deplete their
surroundings with the antibiotic and thus facilitate the growth of the non-resistant ES cells.
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Fig. 3.9: Genetic manipulation of ES cells(A) Shows wild type E14 cells growing on a feeder layer of MEF. (B) Once the ES cells were expanding, they were electroporated with the transgene construct. (C) Depicts the electroporated Neor ES cell colonies. (D) X-Gal staining of the isolated ES cell clones showing the different kinds of the clones obtained.
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3.1.6.2 Characterization of the ES cell clones
For all the ES cells clones isolated, they were expanded on a 24-well plate and
genomic DNA was isolated from them (See 2.2.9.8). GlyT1 specific genotyping PCR was
performed using primer pair 44S/44AS (see Appendix I) and GlyT1 (+) clones were
identified. A representative gel is shown in Fig. 3.10 with GlyT1 (+) ES cell clones showing a
fragment of 497 bp. 300 of the total ES cell clones which were isolated showed a GlyT1∼ ∼
specific band. Correspondingly, all the ES cell clones which were obtained were frozen (See
2.2.9.5) and stored in liquid nitrogen until further use.
Fig. 3.10: Characterization and freezing of ES cells(A) PCR showing ES cell clones positive for GlyT1. (B) The morphology of the GlyT1(+)ES cell clones prior to freezing. Most of the cells showed round morphology, a requisite for undifferentiated ES cell colonies.
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3.1.6.3 Identification of the ES cells carrying a single copy integration of the
transgene: Multicopy PCR
In general, transgenes integrate at random sites in any of the chromosomes of the
genome of host cells. As a result, different cells may be expected to show integration of the
transgene at different chromosomal locations. The number of copies integrated per
genome ranges from one to several hundred. When multiple copies are integrated, they
are mostly integrated at one site joined to each other in either head-to-tail, head to head or
tails to tail fashion, i.e., as a concatemer. In a small proportion of cases, multiple copies can
also be located at several sites in the same genome. However, rarely single copy
integration of the transgene also occurs.
Since the transgene construct was electroporated into the ES cells, which then randomly
integrated into the ES cell genome, it was found imperative to identify the ES cell clones
which carried only a single copy integration of the transgene. This was essential since the
number of copies integrated into the host genome can influence the expression pattern of
the transgene dramatically. Multicopy integrations can give high levels of expression of the
transgene but the expression varies between different animals and a lot of animals need to
be analyzed to get any reliable data. This is circumvented in single copy integrations. A
PCR strategy was designed in a way to distinguish between the clones carrying single copy
and multicopy integrations.
For this PCR, primers were designed in a way which could differentiate between single
copy and multicopy integrates. Two primers were designed in a way that the first primer
binds at the beginning of the construct and the second primer before the first linearization
site. (Fig. 3.11, A). After PCR, different fragment sizes are expected depending upon the
orientation of the transgene and the formed concatemers. For single copy integration, no
PCR product is expected since two primers bind in opposite direction, thereby making no
amplification product (Fig. 3.11, B). The primers will yield a PCR product (products) if
there is/are multicopy integrations, depending upon the orientation of the integrations
(Fig. 3.11, C).
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The different sizes expected for different multicopy integration are as follows:
Heat to Tail 910 bp
Head to Head 1028 bp
Tail to Tail 728 bp
ES cell clones which showed homogeneous LacZ stain were checked for their copy number.
In total, 9 clones were checked with the PCR. ES cell clone 12, 149, and 269 did not show
any amplification product upon PCR analysis. (Fig. 3.11, C). This implied that these clones
probably had single copy integration of the transgene. In contrast, clone 141 showed a
single band at 900 bp (Fig. 3.11, C **), which can be explained by the multicopy∼
integration of the transgene in a head to tail fashion. However, the pattern obtained with
clone 140 was more complex. Apart from showing a band at 900 bp (Fig. 3.11, C **), it also∼
showed a band at 700 bp, which might correspond to a tail to tail integration of the∼
transgene (Fig. 3.11, C ***). From this it can be deduced that this clone carried multicopy
integration of the transgene maybe at different chromosomal locations and in different
orientation. ES cell clones 11, 28, 29, 33 also did not show any band upon PCR amplification
implying that these clones also had single copy integration. However, clones 29 and 33
were further excluded from the study since during expansion, the cells did not show
homogeneous LacZ stain and became undifferentiated upon prolong culture.
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Fig. 3.11: Multicopy PCR: Identification of the clones carrying single copy integration of the transgene(A) Representative diagram of the transgene construct depicting the primer binding sites. The dotted square box indicates the region before the first linearization site. (B) Shows the different types of multicopy integration possible and the product size expected for each configuration. (C) Gel showing different ES cell clones on which the multicopy PCR was performed. The red star (*) indicate clones having single copy integration and then used for further analysis. (**) and (***) indicate bands for the clones which had multicopy integration. The other faint bands which are visible in the gel picture are unspecific bands which were also present in wild type ES cells (data not shown).
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3.1.6.1 Molecular test to check for in vitro cre activation of the transgene: “Non-
activation/Activation PCR”
After ES cell clones containing single copy integration of the transgene were
identified, it was decided to “activate” the transgene in vitro i.e. to excise the silencing
LacZ/Neor cassette upon cre expression. For this, ES cells clones carrying the “silenced
transgene” (i.e. cells electroporated with transgene construct iLacZ/GlyT1-EGFP) and a
single copy integration were expanded on 2*24-well plates. These cells were called as “-
cre-recombined” clones. To induce cre recombination in the expanding ES cells, a transient
expression of a plasmid carrying cre recombinase was carried out. The pGKCre plasmid
(Ref.) was electroporated into half of the expanding ES cells and allowed to express for two
days. pGKCre plasmid contains a bacteriophage P1 recombinase cre with a SV40 large T
antigen nuclear localization signal (NLS-Cre) under a Phosphoglycerate kinase I promoter
(PGK) promoter,which allows the expression of cre recombinase in mammalian cells. The
cells treated with pGKCre were called as “+ cre-recombined” clones. Out of the two plates
containing the expanding clones, one was used for the analysis of the expression of the
transgene using immunocytochemistry as described in the next section. From the other
plate genomic DNA was isolated from the cells and “Non-activation/Activation PCR was
performed on them as described below.
To check for the in-vitro cre recombination in ES cell clones, a PCR strategy was designed to
analyze cre recombination. Three different kinds of primers were designed to distinguish
between “- cre-recombined” and “+ cre-recombined clones. Primer a bind before the first
loxP site in the transgene construct (Fig. 3.12, A, blue arrow), primer b in the silencing
cassette (Fig. 3.12, A, orange arrow) and primer c after the second loxP site (Fig. 3.12, A, red
arrow). PCR which was carried out using primer pair a/b was called as “non-activation”
PCR and that with primer pair a/c as “activation” PCR.
For the non-activation PCR, a product size of 226 bp is expected with the primer pair a/b∼
(representative Fig. 3.12, A). For the activation PCR, a product of 5157 bp and 186 bp is∼
expected with primer pair a/c depending upon the clones (representative Fig. 3.12, A).
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Non-activation PCR was carried out on “- cre-recombined” and “+ cre-recombined” clones.
PCR product of 226 bp was obtained in all “- cre recombined” clones (Fig. 3.12, B, upper∼
gel). A similar band pattern was also observed for the “+ cre recombined” clones (Fig. 3.12,
B, lower gel). This can be explained with the fact that not all the expanding colonies express
cre recombinase but still expressed the silenced transgene construct.
Similarly, activation PCR was also performed on “- cre-recombined” and “+ cre-recombined”
clones. The “- cre-recombined” clones did not show and product since the two primers bind
quite far away from each other and the region in between them could not be amplified (Fig.
3.12, B, upper gel). However, a band of 186 bp was observed for “+ cre recombined”∼
clones (Fig. 3.12, B, lower gel). This proved that cre-recombination occurred in the cells
which were electroporated with cre recombinase. The absence of any band in “- cre-
recombined” lanes shows that the band is specific for cells expressing cre recombinase. The
lack of specific product for clone 11 is an experimental artifact due to less quantity of DNA
used in the PCR.
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Fig.3.12: PCR to check for the activation of the transgene in ES cells(A) Representative diagram of the transgene construct showing the primer binding sites and the product size expected. Primer a is depicted ad blue arrow, primer b as orange and primer c as red arrow. (B) Depicts the PCR results for Non-activation and Activation PCR using primer pairs a/b and a/c respectively. (*) stands for – cre recombined clones and (**) for + cre recombined clones. The positive control used in the PCR is the plasmid ilacZ/ I LacZ/GlyT1-EGFP for the non-activation PCR and I LacZ/GlyT1-EGFP for activationΔ Δ PCR.
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3.1.6.2 X-Gal and DAB staining of the ES cell clones
To substantiate the results obtained from non-activation/activation PCR, X-Gal and DAB
staining was performed on - cre electroporated and + cre electroporated clones to confirm
the in-vitro cre recombination in the clones. For the X-Gal staining, the cells were treated as
described in section (2.2.8.9). DAB staining was performed as described in section (2.2.9.9).
Out of the four clones analyzed for the PCR, clone 12, clone 269, and clone 149 were chosen
for further analysis.
The cells untreated with cre recombinase (- cre recombinase) showed a homogeneous -galβ
expression (Fig. 3.13, A). However, upon expression of cre recombinase (+ cre
recombinase), there is a reduction in the number of cells expressing -gal, although someβ
cells expressing -gal can still be seen (Fig. 3.13, A). This can be explained by the fact thatβ
not all the cells electroporated expressed cre recombinase to allow the recombination to
occur. Hence, the transgene is still silenced in the cells. However, in cells which express cre
recombinase, the cre recombination leads to excision of the LacZ/Neor cassette, thereby not
expressing the -Gal.β
To check for the expression of mycGlyT1 and EGFP upon cre expression, DAB stain using
anti-myc and anti-EGFP antibody was performed. The EFFP expression in ES cells could not
be detected using fluorescence microscopy since these cells have high auto fluorescence.
Both the - cre electroporated and + cre electroporated clones were analyzed using anti
EGFP antibody. The - cre electroporated clones did not show any signal upon detection.
However, cells treated with cre recombinase, showed expression of EGFP as can be seen by
dark brown aggregates in the cell (Fig 3.13, B). This showed that there was an expression of
EGFP upon cre recombination. This also implied that the loxP sites in the transgene
construct were functional. Staining with anti-myc antibody was also tried. However, this
antibody was not sensitive enough to be used for this staining.
Together, the PCR data and the staining confirmed that the transgene construct could be
activated in vitro. Both of the clones were further used for blastocyst injection to generate
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the transgenic mice.
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Fig.3.13: in vitro cre activation of the transgene in ES cells(A) Shows the LacZ staining of the ES cell clones 12 and 149 before and after cre recombinase expression.
(B) DAB staining of the ES cell clones 12 and 149 before and after cre treatment. The cells were stained with anti-EGFP antibody to check for the expression of EGFP. The cells with the dark brown aggregates express EGFP upon cre expression. Scale bar: 50 µm
RESULTS
3.1.7 Production of the chimeric mice by the blastocyst injection of the ES cells
Production of chimeric mice by injecting embryonic stem cells into the cavity of a
mouse blastocyst is a critical step in the generation of mutant mouse models. Following
blastocyst injections, the ES cells become incorporated within the developing inner cell
mass of the embryo and contribute towards different embryonic lineages including the
germline cells which could then help in stable expression of the transgene over generations.
Chimeric mice resulting from the injected blastocysts are composed of tissue derived from
the inner cell mass of the host blastocyst as well as from the ES cells.
ES cell clones 12 and 149 were used for injecting mouse blastocysts for the generation of
the inGlyT1 transgenic mice. 3 days before the blastocyst injections, the ES cells from the
respective clones were thawed and expanded on 6-well plates. A representative picture of
the clones used before the injections is shown in Fig. 3.14, A. The injection of ES cells was
done by Frank Zimmermann from the Centre of Molecular Biology, University of
Heidelberg. In total, 18 blastocysts were injected with for both the clones. The
microinjected blastocysts were transplanted into pseudopregnant female mice. 5 founder
animals were obtained from both the clones (Fig. 3.14, B). For the ES cell clone 12, 2 of the
founders showed > 70% germline transmission. For the clone 149, 1 founder showed >
50% germline transmission. All these 3 founder animals were then mated out with C57/Bl6
animals to establish different inGlyT1 lines.
2 different inGlyT1 mouse lines were established, one from clone 12 (Line 12) and other
from clone 149 (Line 149).
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Fig. 3.14: Production of the chimeras by the blastocyst injection of the ES cells(A) ES cell from clone 12 and 149 during expansion (upper panel) and before blastocyst injection (lower panel). (B) Chimeras obtained after injection.
3.1.7.1 Characterization of inGlyT1 transgenic mice
X-Gal staining of the tail biopsies and PCR were routinely preformed for the
characterization of the inGlyT1 transgenic mice
For the X-Gal staining, a piece of the tail was fixed in 0.2 % glutaraldehyde solution for 5
min. The tail was then washed twice with LacZ washing solution (see 2.1.11) and then
incubated in LacZ staining solution overnight at 37 °C.
Tail samples from animals from both the transgenic lines showed stained tails in contrast to
the wild type tail which did not express the transgene (Fig. 3.15, A). This confirmed that the
transgene was indeed expressing in the offsprings from the founder animals chosen for
breeding.
DNA was also extracted from mouse tails (see 2.2.2.4 and 2.2.2.5) and genotyping PCR was
performed on the samples using primer pair 44S/44AS. Both these primers bind within the
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GlyT1 cDNA and upon amplification produce a product of 497 bp. As can be seen in Fig.∼
3.15, B, animal# 2, 5, and 6 show a band of 497 bp specific to GlyT1 upon amplification. In∼
contrast, animal # 1 and 3 does not show any band which conferred that these animals do
not express the transgene.
Fig. 3.15: X-Gal staining and genotyping on the mouse tails from inGlyT1 transgenic mice(A) X-Gal staining on the tail biopsies from the animals from inGlyT1 mice. (B) Representative gel showing PCR from the DNA samples isolated from tails of inGlyT1 mice.
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3.1.7.2 -gal expression in β inGlyT1 transgenic mice
In order to check for the overall expression pattern of the transgene in the inGlyT1
animals, it was thought to perform whole mount embryo X-Gal staining. For this, E12.5
embryos were dissected from pregnant females and X-gal staining was performed as
described in Cold Spring Har. Protoc.: 2007. In short, 12.5 dpc embryos were dissected from
pregnant females and made free of their extraembryonic membranes in PBS. The embryos
were fixed in a fixing solution (see 2.1.11) at 4 °C for 30-60 min. Embryos were then
washed thrice with washing solution at room temperature for 20 min each and placed in
LacZ staining solution (see2.1.11) till they turned blue. The embryos were later washed
with PBS and serially desiccated in 50 %, 70 %, and 100 % ethanol before taking
photographs (protocol from Black lab, UCSF).
For the further analysis in the study, only the mouse Line #12 was analyzed since there
were some initial breeding problems with the Line #149.
As can be seen in Fig. 3.16, A, the wild-type embryos were colorless upon LacZ staining.
However, embryos expressing the transgene showed a homogeneous LacZ expression in
spinal cord, developing hindbrain, forebrain, eye, heart etc. This meant that the transgene
was ubiquitously expressed in all developing organ types. Fig. 3.16, B shows the magnified
images of the stained spinal cord, developing forebrain and developing hindbrain. In the
developing spinal cord the somites are also stained (Fig. 3.16, A). Stained telecephalic,
diencephalic and midbrain regions were also seen in developing forebrain and the stained
hindbrain (Fig. 3.16, B).
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Fig. 3.16: Expression of the transgene in inGlyT1 transgenic mice(A) LacZ staining of 12.5 dpc embryos. The wild type embryo does not show any LacZ stain. However, the embryo expressing the transgene shows a homogeneous LacZ expression. Legends: fb-forebrain, mb-midbrain, ey-eye, ea-ear placode, np-nasal placode, hb-hindbrain, ht-heart, so-somites, fl-forelimbb, sc-spinal cord, hl-hindlimb. (B) Enlarged view of spinal cord, developing forebrain with different regions and developing hindbrain
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3.1.7.3 Expression of -gal in the brain of β inGlyT1 transgenic mice
To check for the expression of the transgene in brain, LacZ staining was performed
on the brain sections (see2.2.8.7and 2.2.8.8) from the transgenic mice. The mice were first
identified as positives by LacZ staining of the tail and PCR and then their brain further
processed for the staining.
As depicted in Fig. 3.17, A, there was a homogeneous LacZ stain over all the regions of the
brain, showing that the transgene was ubiquitously expressed in all major regions.
However the level of expression in different regions varied. High expression was observed
in the hippocampus, cortex, and the olfactory bulb. Cerebellum on the other hand showed
much lower level of expression (Fig. 3.17, B). The different region of the hippocampus (CA1,
CA2, and CA3) could also be identified (Fig. 3.17, B).
Fig. 3.17: Expression pattern of -gal in brain of β inGlyT1 transgenic mice
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(A) Depicts the LacZ staining on the whole brain section of inGlyT1 transgenic mice. (B) Shows the enlarged images of different stained regions of the brain: Hippocampus, Cortex, Cerebellum, and Olactory bulb. Abb: O: Stratum oriens; P: Stratum pyramidale; R: Stratum radiatum; LM: Stratum lucidum; M: Stratum molecular; G: Stratum granulosum; H: Hilus; DG: Dentate gyrus and CA: cornu ammonis.
3.1.7.4 Transgene expression upon expression of cre recombinase
After the mouse line was checked for expression of the transgene, it was investigated
whether the cre recombinase mediated excision of the LacZ/Neor silencing cassette could
lead to expression of the mycGlyT1 and EGFP upon recombination. For this, transgene
positive animals were mated with Synapsin cre mouse line, where the expression of cre
recombinase is under the control of neuron-specific Synapsin I promoter. Double transgenic
animals i.e animals positive for both GlyT1 and cre (Tg+/cre+) were used for analysis. As a
negative control, animals only positive for GlyT1 but negative for cre were used. For the
analysis, 3 animals from each litter set were used.
In the sections from the double transgenic mice, no EGFP fluorescence was detected upon
checking with fluorescence microscopy (data not shown). Imagining that the level of cre
expression varies from animal to animal and the rate of recombination being low, staining
was performed using anti-EGFP antibody to enhance the sensitivity.
In brain slices from (Tg+/cre+) mice, no EGFP immunreactivity was observed in any of the
brain regions analyzed (Fig. 3.18). The background staining was similar as compared to the
negative control samples. Stainings were also performed using anti-myc antibody.
However, no specific staining was observed in those animals as well (data not shown).
Different matings were also set up with other cre expressing lines such as GFAP cre (Glial
Fibrillary Acid Protein, Glia specific cre) and EmxI cre (forebrain specific cre). However, the
double transgenic animals from these matings too did not show any EGFP fluorescence
(data not shown).
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Fig. 3.18: Immunostaining of the Tg+/Syn cre+ mice showing transgene activationImmunofluorescence in different regions of the brain from Tg+/Syn cre+ double transgenic mice. No EGFP-specific immunreactivity was observed in these mice. The staining is comparable to the negative control, where the transgene is non-activated. The cell nuclei are stained in blue with DAPI. Scale bar: 20 µm.
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3.1.7.5 Transgene expression over generations
Since no activation of the transgene and EGFP fluorescence was observed upon
mating with different cre lines, different reasons were thought which could hamper with
the transgene activation. One reason was there might not be enough cre expression which
could lead to transgene activation. However, with the number of enough number of animals
and cre lines analyzed, this was ruled out. The other probability could be that the robust
expression of the transgene was lost upon generations. To verify this claim, transgenic mice
from four subsequent generations were analyzed for transgene expression by X-Gal
staining. Three sets of animals from different set of litters were analyzed per generation to
rule out any animal to animal variation.
As can be seen in Fig. 3.19, there was a subsequent loss of transgene expression over the
generation. The expression seemed to be robust during the N1 generation, but got
substantially reduced over subsequent generations. Different regions of the brain were
analyzed to also check for region to region variation of the transgene expression. However,
as can be seen in Fig. 3.19, there was loss of expression in most brain regions. There was so
substantial loss of the expression that the expression in the tail by X-Gal staining could also
not be detected reliably (data not shown).
Thus, it can be concluded from these findings that the expression of the transgene reduced
over the generation. Since the cells expressing the transgene reduced dramatically, the
probability of the cells expressing cre got further reduced. This even lowered the
probability expression of both cre and the transgene in the same cells. Thus, it was deduced
that with so less number of cells expressing the transgene in subsequent generation, the
probability of getting cells expressing both transgene and cre is minimal.
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Fig. 3.19: Transgene expression over generationsRepresentative figure showing loss of transgene expression over generations. There was a substantial loss of expression of the transgene in most of the brain regions analyzed from generation N1 to N3.
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RESULTS (PART II)
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3.2 Developmental expression pattern of Glycine transporters
As described previously in this thesis, the neurotransmitter action of glycine is
terminated by the action of two glycine specific transporters: GlyT1 and GlyT2. These two
transporters act synergistically to remove the extracellular glycine from the synaptic cleft
(Eulenburg et. al., 2005). Knockout mice of both GlyT1 and GlyT2 were generated
previously to understand the physiological functions of these two transporters at the
synapses (Gomeza et. al., 2003 a and b). The phenotypes observed in these mice indicated
that early neonatal lethality in the GlyT1-/- mice is caused by the loss of glial GlyT1 in brain
stem and spinal cord. This contrasts with the early postnatal lethality of GlyT2-/- mice (P10-
P15) due to the loss of GlyT2 in the caudal regions of the brain.
Due to the contrasting phenotypes observed in the knockout mice for both the transporters,
it was suggested that the functions of these two transporters might differ in the mature CNS
from those at the neonatal stages. Earlier reports on the expression profiles of the two
transporters in mice showed that the mRNA for GlyT1 is detected as early as E 9 and E l0,
increased significantly at E 13 and remain at high levels up to E 15 (Adams et. al., 1995). In
contrast, the expression of GlyT2 was detected at E 11 which increased by E 15. Also, the
regions of the embryo expressing GlyT2 were quite distinct from those expressing GlyT1
(Adams et. al., 1995). The expression pattern of the two transporters over development also
varies amongst different species. However, no molecular data is available till date to clearly
define the expression pattern of these two transporters over different stages of
development. The second part of the study described in this thesis, discuss the expression
pattern and role of the two glycine transporters over development.
To check for the expression pattern of these two transporters over development,
membrane preparations were prepared from different aged C57/Bl6 mice. Glycine uptake
experiments and western blots were performed on the membrane preparations and the
expression pattern of these two transporters was analyzed.
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3.2.1
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Appendix I: List of oligonucleotides used in this study
Primer Oligosequence (5’-3’) Features
44S AGG CGT GGG CTA TGG TAT GAT G Genotyping GlyT1 Tg mice
44AS GAA CAA ACA GAA TGG TCA GCA CC Genotyping GlyT1 Tg mice
45S TGT GGA TGA GGT AGG GAA TGA GTG Genotyping GlyT1 Tg mice
45AS ACT GGT AGT GGT TGT AGG TGA TTG G Genotyping GlyT1 Tg mice
CS 26S ACC AGC CAG CTA CTA TCA ACT C Cre recombinase, genotyping
CS 26AS TAT ACG CGT GCT AGC GAA GAT CTC CAT
CTT CCA GCA G
Cre recombinase, genotyping
Cre a1 TAC AGC TCC TGG GCA ACG TG Non activation PCR, binds in the promoter region
Cre b1 AAC GCC AGG GTT TTC CCA Non activation PCR, binds in the LacZ region
Cre c4 CAA CAT CCC TTT GGC ACC Activation PCR, binds in the myc-GlyT1 start region
mcf ScaI TTC CTC CTC TCC TGA CTA CTC Multicopy PCR forward
mcb ScaI CGC ATA CAC TAT TCT CA GAA Multicopy PCR backward
YFP4 GAC CAC TAC CAG CAG AAC AC Sequencing EGFP
PolyA b2 CAG CTA TGA CTG GGA GTA G Sequencing poly A tail
LoxP F1
CS 1S GTC GGA TCC GCA CTG AAC GCA AGA GTC TG
Sequencing plasmid
CS 1AS CTC CTC GGA CAT CAG CTT CTG CTC CAA CAT CCC TTT GGC ACC
Sequencing plasmid
CS 2S CAG AAG CTG ATC TCC GAG GAG GAC CTG AAT GGT GCT GTC CCC AGC
Sequencing plasmid
CS 4AS CAG CTC GAG TCA GGA ACA CTG GTC ACG AG
Sequencing plasmid
All oligonucleotides were obtained from Metabion (Berlin, Deutschland).
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Appendix II: Description of primer pairs used in this study and the size of
the amplified product obtained
Primer pair Purpose Annealing temp
Polymerase Product size
44S/44AS Genotyping inGlyT1 Tg mice
68 °C PAN scriptTM
polymerase497 bp∼
26S/26AS Genotyping Cre mice 56 °C Qiagen hotstart polymerase mix
300 bp∼
Cre a1/b1 Activation/Non-activation PCR
Qiagen hotstart polymerase mix
Cre a1/c4 Activation/Non-activation PCR
Qiagen hotstart polymerase mix
Mcf ScaI/Mcb ScaI
Multicopy PCR Qiagen hotstart polymerase mix
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