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Combined thesis 1

  1. 1. Analysis of in vivo Glycine transporter 1 functions by transgenic approaches<br />Dissertation<br />zur Erlangung des Doktorgrades<br />der Naturwissenschaften<br />vorgelegt beim Fachbereich 14<br />Biochemie, Chemie und Pharmazie<br />der Johann Wolfgang Goethe-Universität<br />in Frankfurt am Main<br />von<br />Deepti Lall<br />aus Delhi (India)<br />Frankfurt 2011<br />(D30)<br />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.<br />Dekan: Prof. Dr. Dieter Steinhilber<br />1. Gutachter: Prof. Dr. Alexander Gottschalk<br />2. Gutachter: Prof. Dr. Heinrich Betz<br />Datum der Disputation:<br />A father's goodness is higher than the mountain,<br />A mother's goodness deeper than the sea.<br />For Mumma and Papa<br />Introduction<br />Inhibitory neurotransmission in the CNS<br />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.<br />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.<br />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.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).<br />MATERIALS AND METHODS<br />MATERIALS<br />Animals, Cell lines and Bacterial strains<br />Table 2-1: List of strains used in the study<br />StrainSourceMouse (Mus musculus, C57/Black6)Charles River, Sulzfeld, GermanySynapsin cre mouse lineZhu et. al. (2001)GFAP cre mouse lineMarino et. al (2000)Emx cre mouse lineSlezak et. al (2008)Escherichia coli XL1-BlueStratageneTM (Amsterdam, Netherland)E. coli DH5-InvitrogenTM (Carlsbad, USA)Sv129/OlaHasd (E14TG2A) ES cell lineUlrike Mueller, Max Planck Institute for Brain Research, Frankfurt.MTK-neo CD1 transgenic miceref<br />Chemicals and plastic materials<br />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).<br />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).<br />Enzymes & others<br />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).<br />DNA standard<br />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. <br />Protein standard<br />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). <br />Membranes and films<br />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.<br />Commercial Kits for molecular biology <br />For standard molecular biology experiments, commercial kits were employed and all the protocols were performed according to the manufacturer’s instructions.<br />Table 2-2: Commonly used kits.<br />NameDescriptionCompanyPlasmid kits(Mini, Midi, Maxi)Purification of plasmid DNAQiagen, (Hilden, Germany)QIAquick Gel extraction kitExtraction of DNA from agarose gelsQiagenPCR purification kitPurification of PCR productsQiagenSuperSignal Western Blotting chemiluminescent substrate kitEnhanced 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 membraneStratagene,TM<br />Culture media and solutions <br />The following list of culture media and solutions were used during various cell-biological and biochemical studies.<br />Bacterial Culture medium<br />Table 2-3: Culture medium used for growing bacteria<br />NameCompositionLB medium1 % (w/v) NaCl, pH 7.5, 1 % (w/v) peptone , 0.5 % (w/v) yeast extractLB AgarLB medium with 1.5 % (w/v) agar<br />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<br />Antibiotics<br />Table 2-4: Antibiotics used for bacterial culture<br />AntibioticStock solutionFinal concentrationAmpicillin100 mg/ml in water50 µg/mlKanamycin50 mg/ml in water50 µg/mlTetracycline10 mg/ml in 50 % ethanol12.5 µg/ml<br />All antibiotics were sterile-filtered and stored at -20 °C.<br />Cell culture media for mammalian cells<br />Table 2-5: Cell culture media<br />NameCompositionDMEM medium +++ 10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/μg/ml), 2 mM L-Glutamine in 500 ml DMEM (Invitrogen).Freezing medium500 ml DMEM medium +++, 10 % (v/v) DMSOGelatin0.5 % Gelatin in sterile distilled H2O100 X poly-D-ornithine150 μg/ml, Poly-D-ornithine-hydrobromide in sterile distilled H2O100 X poly-D-lysine 250 µg/ml Poly-D Lysine in distilled H2O<br />Medium and supplements for ES and MEF cell culture<br />Table 2-6: Cell culture media for ES and MEF cells<br />NameCompositionCompanyMEF mediumDMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep stock (5000 U/µg/ml), 2 mM L-GlutamineDMEM and FCS Gibco-BRL 2X MEF freezing mediumMEF medium, 20 % (v/v) DMSOES cell mediumDMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/µg/ml), 2 mM L-Glutamine,10 mM NEAA, 0.1 mM β-Mercaptoethanol, 1000 U/ml LIFLIF (10,000 U/ml) :Life Technologies, (New York, USA).ES cell selection mediumES cell medium, 1000 U/ml LIF, 0.2 mg/ml GeneticinGeneticin :InvitrogenES cell freezing mediumES cell medium, 50 % (v/v) DMSO<br />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.<br />Electroporation of ES cells<br />For the electroporation of the ES cells the following media was used.<br />Table 2-7: Electroporation medium for the embryonic stem cells<br />NameComposition10X HBSS1X1 M HEPES 20 mMβ-Mercaptoethanol0.1 M1M NaOH1 mM<br />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).<br />General buffers and solutions<br />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.<br />Table 2-8: Solutions for molecular biology<br />NameComposition10X PBS80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, pH 7.3PMSF100 mM in isopropanol10X DNA loading buffer20 % (v/v) Ficoll 400, 0.1 mM EDTA, 1 % (w/v) SDS, 0,25 % (w/v) Bromophenolblue, 0,25 % (w/v) Xylene Cyanol FF20X SSC buffer3.0 M NaCl, 0.3 M Trisodium citrate pH 7.050X Denhardt’s solution1 g BSA, 1 g PVP, 1 g Ficoll50X TAE242 g Tris base, 57.1 ml glacial acetic acid, 37.2 g Na2EDTA.2H2O, pH∼8.5Brain P2 prep membrane preparation medium0.33 M sucrose, 1 mM EDTA, 1 mM PMSF, 10 mM HEPES-Tris pH 7.4DNA extraction “salting out” buffer200 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 PI50 mM Tris-Cl, 10 mM EDTA pH 8.0, RNase A (100 µg/ml)DNA isolation Buffer P2200 mM NaOH, 1 %(w/v) SDSDNA isolation Buffer P33 M Potassium acetate, pH 5.5HEK293 cell lysis buffer150 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 medium125 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.3 mM MgSO4, 10 mM Glucose, 25 mM HEPES-Tris pH 7.4LacZ stain washing buffer2 mM MgCl2, 0.01 % (w/v) sodium deoxycholate, 0.02 % (v/v) NP-40 in 1X PBS pH 7.3LacZ staining buffer5 mM Potassiumferrocyanide, 5 mM Potassiumferricyanide, 0.5 mg/ml X-Gal (in DMF) in 1X PBS pH 7.3LacZ fixative for embryo staining1 % formaldehyde, 0.2 % glutaraldehyde, 5 mM EGTA, 0.02 % NP-40 in 1X PBSLacZ staining buffer for embryo staining5 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 7.3Lysis buffer ES cells for PCR100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 0.2 M NaClLysis buffer for ES cells100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 200 mM NaCl, 62.5 µg/ml Proteinase KMouse tail lysis buffer20 mM NaCl, 10 mM Tris-Cl ph 8.5, 5 mM EDTA, 0.1 % (w/v) SDS , 210 µg/ml Proteinase K (25 µg/µl stock solution)PBS + Tween0.05 % Tween-20 in 1X PBSSouthern blot denaturation buffer0.5 M NaOH, 1.5 M NaClSouthern blot hybridisation solution50 ml 50X Denhardt’s, 125 ml 20X SSC, 25 ml 10 % (w/v) SDS, 5 ml of 10 mg/ml denatured salmon sperm DNASouthern blot neutralization buffer5 M NaCl, 0.5 M Tris-Cl pH 7.4Southern blot transfer buffer10 X SSCSouthern blot wash buffer 12X SSC, 0.5 % (w/v) SDSSouthern blot wash buffer 20.2X SSC, 0.5 % (w/v) SDSTE buffer10 mM Tris, 1 mM EDTA<br />Solutions for protein biochemistry<br />Table 2-9: Composition of solutions<br />NameComposition10X Tris-Glycine buffer30.2 g Tris base, 144 g glycine4X Laemmeli buffer8-11 % (w/v) SDS, 4 % (v/v) glycerin, 0.04 % (w/v) Pyronin Y, 20 % (v/v) β-mercaptoethanol, 250 mM Tris-Cl pH 6.8Blocking solution for western blotting5 % (w/v) skim milk powder, 0.05 % (v/v) Tween-20 in 1X PBSPonceau-staining solution3 % (w/v) Glacial acetic acid, 0.3 % (w/v) Ponceau SSDS PAGE running buffer1X TG buffer, 0.1 % SDS, pH 8.8Stripping 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 waterWestern blot transfer buffer20 % Methanol, 0.01 % SDS in 1X TG buffer, pH 8.8<br />Solutions for immunocytochemistry and immunohistochemistry<br />Table: 2.10: Composition of solutions<br />NameComposition2-4% PFA solutionAdd 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 solution1 % (v/v) goat serum, 2.5 % BSA, 0.1-0.3 % Triton-X 100 in 1X PBS pH 7.5Quenching solution100 mM glycine in 1X PBS pH 7.5Quenching solution for DAB staining0.3-1 % H2O2 in 1X PBS pH 7.5<br />Vectors<br />Table 2-11: List of vectors used (in the study)<br />NameOrigin/ ReferenceAntibiotic resistancepBluescript II SK (+)Stratagene (Heidelberg, Germany)AmpicillinpBK CMVStratagene (Heidelberg, Germany)KanamycinpcDNA3.1Invitrogen, (Carlsbad, USA)AmpicillinpcDNA3.1/ DLC mycPetra Scholze, MPIH, FrankfurtAmpicillinpCCALL2-IRES-EGFP/antonLobe (1999)AmpicillinpCCALL2-IRES-EGFP/anton/GlyT1Chigusa Shimizu Okabe (this study)AmpicillinpEGFP-C3Clontech (Heidelberg, Germany)AmpicillinpPGKcrebpAKlaus Rajewsky, (Harvard Medical School, Boston, MA, U.S.A.)Ampicillin<br />Antibodies<br />Table 2-12: List of primary antibodies used (in the study)<br />NameAntigenSpeciesDilutionWBICC/IHCSourcemAb4a hybridoma supernatantAS 96-105 from GlyRα1mouse Dr. Carmen Villman, Uni-ErlangenGFPGFP isolated directly from Aequorea victoriarabbit 1:1000 1:500InvitrogenVIAATAS 75-87 from VIAATrabbit1:1000Synaptic Systems, Göttingenmyc 9106EQKLISEENLrabbit 1:200Abcam, Cambridge, U.Kβ-Gal 4761Full length native protein (purified from E. coli).rabbit 1:250Abcam, Cambridge, U.KGlyT1(M7/M14)rabbit1:1000Eulenburg, 2010GlyT2NN terminal cytoplasmic domain of rat GlyT2rabbit1:2000Gomeza, 2003aGRP75 (JG1) 2799AS 615-633 of Mouse Grp75mouse1:1000Abcam, Cambridge, U.K<br />Table 2-13: Fluorescent conjugated secondary antibodies used for ICC and IHC<br />NameDilutionSource/ReferenceAlexa 488-anti-mouse1:500Molecular Probes (Eugene, USA)Alexa 546-anti-mouse1:500Molecular Probes (Eugene, USA)Alexa 488-anti-rabbit1:500Molecular Probes (Eugene, USA)Alexa 546-anti-rabbit1:500Molecular Probes (Eugene, USA)<br />Table 2-14: Peroxidase-linked secondary antibodies for western blot<br />NameDilutionSource/ReferenceHRP-anti-mouse1:10000Dianova (Hamburg, Germany)HRP-anti-rabbit1:10000Dianova (Hamburg, Germany)<br />METHODS<br />Biochemistry and molecular biology methods<br />Growth conditions and culture methods for microbial culture<br />The microbial cultures were grown under standard conditions as described 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ADDIN EN.CITE 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ADDIN EN.CITE.DATA (Bertani, 1951; Rose, 1990; Sambrook, 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 REF _Ref282093761 r h<br />Preparation of E. coli chemical competent cells<br />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. <br />Preparation of E. coli electrocompetent cells<br />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 OD600 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. <br />Preparation of bacterial glycerol stocks<br />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.<br />Heat shock transformation of plasmid DNA<br />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 ( 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 ( REF _Ref282166918 r h containing the appropriate antibiotic and incubated O/N at 37 °C.<br />Electroporation<br />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 ( REF _Ref282166918 r h 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.<br />Isolation and purification of plasmid DNA <br />Small-scale purification of plasmid DNA <br />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.<br />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.<br />Large scale plasmid DNA preparations ("midi/maxi preps")<br />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.<br /> DNA extraction from mouse tissue<br />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 ( REF _Ref282179248 r h 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 ( REF _Ref282179248 r h 2.1.11) was added to the samples. The samples were incubated O/N at 56 °C and 800 rpm.<br />Phenol-chloroform extraction of the genomic DNA <br />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 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 ( REF _Ref282179248 r h 2.1.11).<br />Genomic DNA isolation by “salting out”<br />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 ( REF _Ref282179248 r h 2.1.11).<br />Analysis of the purified DNA<br />Agarose gel electrophoresis for the separation of the DNA fragments<br />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 μg/ml) was added to it. The warm agarose was slowly poured into a gel tank and combs were inserted. The set was kept undisturbed till the agarose polymerized.<br />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 ( REF _Ref287359070 r h 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.<br />Determination of DNA concentration by spectrophotometry<br />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). <br />For RNA: A260/A280=2.0, <br />DNA: A260/A280=1.8 and <br />Protein: A260/A280=0.6.<br />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).<br />Extraction of DNA from agarose gels<br />To isolate the desired DNA fragments from a sample after restriction digestion or PCR amplification, it was subjected to a gel electrophoresis ( 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 ( REF _Ref287359794 r h 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.<br />DNA-sequencing<br />The separation, detection, and sequencing, of DNA products were performed by the service provider MWG Biotech. Inc. (Ebersberg, Germany).<br />Enzymatic modification and manipulation of the DNA<br />Restriction digest of the DNA<br />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 μg of DNA in 1 hour at 37 °C. For analytical DNA digests, usually 500 ng-10 μg (for southern blot) of the 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 sensitive enzymes) and the DNA fragments were isolated after electrophoretic separation in an agarose gel using QIAQuick gel extraction kit.<br />Dephosphorylation of the DNA digested with restriction<br />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 1μM p-nitrophenolphosphate to nitrophenol in 1 min at 37 °C. To remove 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, ( REF _Ref287359794 r h 2.1.7).<br />Ligation of DNA fragments<br />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 μl reaction volume and a 5’ termini concentration of 0.12 μM (300 μg/ml).<br />DNA fragments were ligated by mixing 25-50 ng vector DNA with a threefold excess of insert DNA. 0.5 μl of T4-ligase and 1 μl of 10 X ligation buffer were added and the reaction mixture was brought to a final volume of 10 μl with ddH2O. The reaction was incubated O/N at 16 °C. The reaction mixture was used directly for transformation without any further purification.<br />DNA amplification: Polymerase chain reaction<br />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.<br />Random-labeling of DNA probes<br />DNA probes were radioactively labeled with α-[P32]-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.<br />Table 2-15: Reaction mix for random labeling of DNA probes<br />ComponentAmountTemplate DNA9 µl (10 ng – 3 µg)dTTP1 µldATP1 µldGTP1 µlHexanucleotide buffer2 µlα-[P32]-dCTP5 µlKlenow enzyme 1.5 µl<br />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 ( REF _Ref282179248 r h 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.<br />Southern blot analysis of genomic DNA <br />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 ( REF _Ref282179248 r h 2.1.11) and then transferred using neutral transfer, O/N onto a nylon membrane ( REF _Ref129948984 r h 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.<br />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 ( REF _Ref129948984 r h 2.1.6) with intensifying screens and developed after 3 days <br />Table 2-16: Hybridisation solution for Southern blot<br />ComponentAmountFormamide20 mlSalmon sperm DNA*1 ml1M NaH2PO4, pH 6.52 ml10 % SDS2 ml50X Denhardt’s sol4 ml0.5 M EDTA, pH 8.00.4 ml20X SSC12 ml<br />*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.<br />Brain total RNA preparation <br />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.<br />First strand c-DNA synthesis<br />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.<br />In brief, 50 pmol of oligo-dT primer and 1 μg total RNA were added to a nuclease-free microfuge tube. The mixture was heated at 65 °C for 10 min. 4 μl of 5 X First-Strand Buffer, 0.5 μl of Protector RNase inhibitor, 2 μl of 10 mM dNTP’s and 0.5 μl of reverse transcriptase were added to the tubes and final volume of the mix was adjusted to 20 μl 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 μl (2 units) of E. coli RNase H at 37 °C for 20 min to remove >1kb RNA. <br />Cell biology methods<br />Coating of coverslips and culture plates with poly-D-ornithine and poly-D-lysine<br />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.<br />Culture and maintenance of HEK 293T cells<br />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 ( REF _Ref287368197 r h 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.<br />Freezing and thawing of cell lines<br />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 ( REF _Ref287368534 r h 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.<br />To revitalize frozen cells, a very rapid warming up of the tube in a water bath at 37 °C was carried out. The thawed aliquot was mixed with 10 ml of prewarmed culture medium ( REF _Ref287368534 r h and distributed in 10 cm culture dishes. The cells were grown and passaged as explained above in REF _Ref287368828 r h<br />Transfection of HEK 293T cells using Lipofectamine 2000TM<br />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.<br />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 ( REF _Ref282093761 r h 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.<br />Preparation of lysates from HEK 293T cells<br />After 24-hr expression by HEK 293T cells, the cells were washed twice with PBS and 500 µl of HEK 293T cell lysis medium ( REF _Ref282179248 r h 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.<br />Immunocytochemistry on HEK 293T cells<br />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 ( REF _Ref287370777 r h 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 REF _Ref287370972 r h 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 ( REF _Ref287370972 r h 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.<br />Cryostat brain sectioning<br />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.<br />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 ( REF _Ref287370777 r h 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.<br />Immunostaining on brain sections<br />For immunostaining, tissue slides were dried at room temperature and fixed for 10 min in ice-cold 4 % PFA ( REF _Ref287370777 r h 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 ( REF _Ref287370777 r h 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 ( REF _Ref287370777 r h 2.1.13) for 2 hr at room temperature. The slides were incubated in primary antibody (appropriate dilutions in REF _Ref287370972 r h 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 ( REF _Ref287370972 r h 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).<br />Detection of β-galactosidase in tissue sections (X-Gal staining)<br />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).<br />To detect β-galactosidase activity in transgenic mice, slides containing 14-μm vibratome sections were washed in LacZ washing solution ( REF _Ref282179248 r h 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 ( REF _Ref282179248 r h 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.<br />Confocal microscopy, image acquisition and analysis <br />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).<br />Embryonic stem cell (ES) culture<br />Culture of Embryonic stem cells<br />ES cells were routinely grown on a monolayer of MEF cells at 37 °C in a 5 % CO2 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 ( REF _Ref287368197 r h 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. <br />Thawing of ES cells<br />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.<br />Preparation of mouse embryonic fibroblasts (MEF’s)<br />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.<br />Feeder irradiation<br />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.<br />Freezing of cells<br />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 ( REF _Ref287368197 r h 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.<br />Electroporation of the transgene construct into ES cells<br />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 ( REF _Ref287376114 r h 2.1.10). All steps were performed under sterile conditions using sterile consumables under the hood.<br />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, τ = 5.7-6.0 ms. The cells were 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 the selection medium for 5-7 days. The medium was exchanged regularly to allow proper growth of the cells.<br />Selection of ES cell clones<br />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.<br />Preparation of DNA from ES cells<br />500 µl of ES cell lysis buffer ( REF _Ref287368197 r h 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.<br />DAB staining of the ES cells<br />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 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.<br />Preparation of ES cell clones for blastocyst injection<br />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 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.<br />Glycine uptake experiments<br />Brain membrane preparation<br />Mouse brain samples were homogenized in 1 ml of ice-cold P2 prep membrane preparation medium ( REF _Ref282179248 r h 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 ( REF _Ref282179248 r h 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).<br />(3H) Glycine Transport Assay<br />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<br /> Relative uptake = cpm/ [total glycine concentration] (pmol)<br /> Total protein conc. (mg) * time (min)<br />Protein biochemistry methods<br />Protein determination by Bradford assay<br />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. <br />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.<br />Discontinuous SDS Polyacrylamide Gel Electrophoresis<br />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 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.<br />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.<br />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 ( REF _Ref287381269 r h 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.<br />Western blot analysis<br />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 ( REF _Ref287382457 r h 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.<br />Following electrophoresis, the separated proteins were transferred from the SDS polyacrylamide gel to the membrane ( REF _Ref129948984 r h 2.1.6) by electrotransfer using a Mini-Trans-Blot electrophoretic chamber (Bio-Rad; Munich, Germany). The transfer was carried out in transfer buffer ( REF _Ref287370770 r h 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.<br />Immunodetection of blotted proteins<br />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 ( REF _Ref287370770 r h 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 ( REF _Ref287370972 r h 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 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).<br />RESULTS (PART I)<br />Role of Glycine and Glycine transporters in the CNS<br />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, 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, 1995 a,b).<br />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, 2008). The simultaneous activation of excitatory NMDAR’s and inhibitory GlyR’s may provide a homeostatic regulation of hippocampal network function. <br />In the hippocampus, GlyT1 is mainly expressed by astrocytes and neuronal GlyT1 is only found at glutamatergic synapses (Cubelos, 2005). At these synapses, it is postulated that astrocytes via GlyT1 are the major source of hippocampal glycine (Yang, 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.<br />Considerations for construct design for generation of an inducible (in)GlyT1 transgenic mice<br />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’.<br />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 β-actin promoter are 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.<br />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.<br />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.<br />“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.<br /><ul><li>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, 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, 2001). This makes this system unsuitable for the investigation of acute transgene effects.
  2. 2. 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.”</li></ul>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. <br />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 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).<br />Fig. 3.1: Strategy for the generation of inGlyT1 transgenic mice<br />The 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. <br />Generation of the transgene construct<br />The expression vector used for the generation of the inGlyT1 mouse line was based on an approach from Lobe, 1999 and {Please_Select_Citation_From_Mendeley_Desktop}Novak, 2000 The expression of the transgene is driven by ubiquitously active chicken β-actin promoter. An enhancer element cassette (CAG cassette) from Cytomegalovirus (CMV) (Xhu, 2001, Niwa, 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.<br />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, 1994) and GlyT1. This construct was designated as iLacZ/GlyT1-EGFP.<br />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.<br />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 iΔLacZ/GlyT1-EGFP Characterization of tagged GlyT1 constructs<br />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.<br />Glycine uptake experiments were performed after 2 days as described in section REF _Ref286339267 r h 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.<br />For western blot analysis, lysates from HEK 293 cells transfected with different constructs were prepared as described in section 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.<br />Fig 3.3: Characterization of the tagged GlyT1 constructs<br />(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).<br />Verification of the transgene construct<br />After the generation of the transgenic construct, different regions of it were verified using PCR, restriction digest analysis and sequencing. <br />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.<br />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).<br />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. <br />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 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.<br />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.<br />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).<br />Fig. 3.4: Validation of the transgene construct<br />The 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.<br />Heterologous expression of the transgene construct in HEK 293 cells<br />After the transgene constructs was generated and verified, their functionality was checked in HEK 293 cells. For the experiments, iLacZ/GlyT1-EGFP and IΔLacZ/GlyT1-EGFP plasmid DNA were transfected into HEK 293 cells (see REF _Ref288155844 r h and the expression of the markers, β-gal and EGFP, were analyzed after 2 days of expression.<br />To check for the cells expressing β-gal encoaded by the LacZ gene in the construct, X-Gal 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 β-galactosidase. X-gal is cleaved by β-galactosidase yielding galactose and 5-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.<br />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 β-gal (Fig. 3.5, B1). In the same cells no EGFP fluorescence was detected since the expression of EGFP was silenced (Fig. 3.5, B2). Cells transfected with IΔLacZ/GlyT1-EGFP did not show any β-gal 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.<br />Fig. 3.5: Functionality test of the constructs in HEK 293 cells<br />To check for the functionality of the constructs, HEK cells were transfected with the indicated plasmids. Expression of β-gal or EGFP was analyzed by X-Gal assay or fluorescence microscopy. (A1, A2): untransfected HEK cells stained for β-gal 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 IΔLacZ/GlyT1-EGFP , lacked β-gal expression but show EGFP fluorescence with the soluble EGFP accumulated all over the cell. Scale bar: 50µm<br />Immunostaining with different antibodies was also performed on fixed and permeabilized transfected HEK 293 cells to check for the expression of the markers β-gal, EGFP, and mycGlyT1. Different dilutions of the antibodies which were used are listed in section REF _Ref287370972 r h 2.1.15.<br />To check for the expression of β-gal, immunostaining with rabbit anti- β-gal and Alexa 546 antibody was done. EGFP fluorescence was analyzed by fluorescence microscopy. The 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 β-gal localized both in the cytoplasm as well as in the cell membrane (Fig. 3.6, 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 β-gal is expressed and the expression of EGFP is silenced due to the presence of LacZ/Neor silencing cassette. However, cells transfected with plasmid IΔLacZ/GlyT1-EGFP did not show any β-gal 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).<br />Fig. 3.6: Immunostaining of HEK 293cells with anti β-Gal antibody<br />Expression of the markers β-gal and EGFP upon staining with anti β-gal antibody. (A1-C1) Show untransfected HEK 293 cells; (A2-C2) and (A3-C3) depict staining of HEK 293 cells expressing iLacZ/GlyT1-EGFP and IΔLacZ/GlyT1-EGFP respectively. (B1-B3): depict the cell nuclei co-stained with DAPI. Scale bar: 50 µm.<br />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 REF _Ref287370972 r h 2.1.15). EGFP fluorescence was analyzed by fluorescence microscopy as described previously.<br />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 IΔLacZ/GlyT1-EGFP show expression of mycGlyT1 as seen by the 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 IΔLacZ/GlyT1-EGFP.<br />Fig. 3.7: Detection of mycGlyT1 and EGFP<br />Fluorescence images showing HEK 293 cells transfected with iLacZ/GlyT1-EGFP and IΔLacZ/GlyT1-EGFP 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 IΔLacZ/GlyT1-EGFP 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.<br />Backbone removal and linearization of construct<br />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 <br />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).<br />Fig. 3.8: Backbone removal and linearization of the plasmid iLacZ/GlyT1-EGFP<br />(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