This document describes the materials and methods used in a dissertation analyzing the in vivo functions of Glycine transporter 1 (GlyT1) through transgenic approaches. It provides details on mouse strains, cell lines, bacterial strains, chemicals, enzymes, kits, culture media, buffers and solutions used for experiments involving molecular biology techniques, cell culture, protein biochemistry, and transgenic mouse models. The goal was to generate and characterize GlyT1 transgenic mouse lines to study the role of GlyT1 in inhibitory neurotransmission.

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.”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 et.al., 1999 and {Please_Select_Citation_From_Mendeley_Desktop}Novak et.al., 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 et.al., 2001, Niwa et.al., 1991) is cloned upstream of the promoter to direct strong expression. Expression of the transgene GlyT1 is prevented by the expression of a silencing cassette containing a first reporter, a LacZ/Neor fusion protein; (Friedrich and Soriano, 1991) that was followed by a triple repeat of the simian virus (SV40) polyadenylation signal. The reporter along with the stop signal is flanked by two loxP sites. This allows the removal of the silencing cassette upon expression of cre recombinase (Fig. 3.2, B). The original vector map is listed in appendix III.<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 et.al., 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 2.2.10.2. The glycine uptake experiments revealed no significant difference between the uptake activities of cells transfected with tagged and untagged GlyT1 constructs. Both tagged constructs showed a concentration-dependent uptake of radiolabeled glycine, although GlyT1-N showed much less uptake activity than GlyT1-C. Untransfected HEK 293 cells and cells transfected only with pcDNA3.1 also showed minimal glycine uptake activity which was much less than cells expressing the transporter constructs (Fig.3.3, B). Thus, it can be concluded that the tagged transporter constructs were functional and can transport glycine.<br />For western blot analysis, lysates from HEK 293 cells transfected with different constructs were prepared as described in section 2.2.8.5 and detected using anti-myc and anti-GlyT1 antibodies (Fig. 3.3, C). The cells expressing both myc-tagged constructs were recognized by anti-myc and anti-GlyT1 antibodies respectively. When probed with anti-myc antibody, immunoreactivity was detected for GlyT1-N as well as GlyT1-C whereas the untransfected and cells expressing wildtype GlyT1 didn’t show any immunreactive bands. On probing with anti-GlyT1 antibody, cells expressing both myc tagged constructs and endogenous GlyT1 were recognized. The different band sizes at 110 kDa, 98 kDa and 58 kDa depict the different complex glycosylated and unglycosylated forms of GlyT1 (Fig. 3.3, C). Since N-terminally tagged construct showed glycine dependent uptake activity and was more reliably detected in the western blots, it was further chosen for the generation of inGlyT1 transgenic mice.<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 2.2.8.4) 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 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 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 plasmid iLacZ/GlyT1-EGFP with SfiI and Eam1105 I.<br />Genetic manipulation of the Embryonic stem (ES) cells<br />For the generation of the transgenic mice, the E14