Generation of a CKO targeting vector

The aim was to generate a mouse in which exon 21 in the Munc13-1 gene is flanked by two loxP sites, so that upon site-specific Cre-recombinase activity in the neurons exon 21 will be deleted. This, in turn, is predicted to result in a frame shift in the gene transcription product, and therefore to a loss of the Munc13-1 protein.

For the generation of a conditional knockout (CKO) targeting vector, I chose recombination-mediated genetic engineering (recombineering), a method based on homologous recombination via gap repair in E.coli bacterial cells. This method utilizes the specialized E.coli strains E350 and SW106 (Lee et al., 2001; Liu et al., 2003; Warming et al., 2005), derived from DH10B BAC DNA host cells and modified to express enzymes required for gap repair (Exo, Beta, and Gam) in a temperature-dependent manner from a

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cyclization recombination (Cre)-recombinase under an L-arabinose-inducible promoter.

E350 and SW106 strains only differed by an additional galK-based selection system integrated into SW106, which I did not employ in this study (Warming et al., 2005). The expression of these enzymes enables recombination of DNA fragments between vectors carrying homologous sequences.

Figure 3.20. Generation of a Munc13-1 conditional knock-out targeting vector

Schematic representation of the cloning strategy for a Munc13-1 conditional knock-out (cko) targeting vector. Exons are labeled as grey boxes, introns as white boxes and exon 21 as a red box. White triangles indicate loxP sites, black triangles indicate FRT sites. 1. BAC DNA retrieval via gap repair: Two homology arms (A+B) were amplified by PCR and subcloned into the PL253 retrieval vector. The vector was linearized by HindIII digest. A 9.7 kb fragment of the Munc13-1 gene containing exon 21 was retrieved from the SV129 embryonic stem (ES) cell derived bacterial artificial chromosome (BAC) plasmid bMQ411l13 via homologous recombination into the PL253 retrieval vector. 2. Targeting of the first loxP site: The two homology arms (C+D) were amplified by PCR and subcloned into the PL452 vector plasmid to flank a floxed neomycin resistance gene (Neo) cassette. The floxed Neo cassette with the two homology arms was excised by KpnI and NotI digestion, together with ScaI to cut the plasmid backbone in the Amp gene. The floxed Neo cassette was inserted upstream of exon 21 via homologous recombination and excised after L-arabinose-induced Cre-recombinase expression, resulting in a single loxP site with a diagnostic HindIII restriction site. 3. Targeting of the second loxP site: The two homology arms (E+F) were amplified by PCR and subcloned into the PL451 vector plasmid to flank a Neo cassette flanked by FRT sites and a downstream loxP site. The FRT site flanked Neo cassette with the two homology arms was excised by XhoI and SacII digest, together with ScaI to cut the plasmid backbone in the Amp gene, and inserted downstream of exon 21 via homologous recombination. The Neo cassette, HSV-TK cassette, and FRT and loxP sites are not drawn to scale. Neo, neomycin resistance gene;

HSV-TK, herpes simplex virus thymidine kinase; Amp, Ampicilin. Based on (Liu et al., 2003)

Using recombineering, I retrieved a 9.7 kb Munc1 gene fragment containing exons 13-26. This fragment was retrieved from a BAC, namely the 129SV ES cell derived BAC clone bMQ 441l13 containing the full Munc13-1 genomic sequence. bMQ 441l13 is part of a 129SV ES cell DNA BAC library and contains a pBACe3.6 vector backbone with a chloramphenicol resistance gene for selection (Adams et al., 2005; Frengen et al., 1999).

The fragment was retrieved into a plasmid containing a MC1 promotor controlled herpes simplex virus thymidine kinase cassette (MC1TK). I subsequently introduced loxP sites and a neomycin resistance (Neo) cassette for positive selection in embryonic stem (ES) cells (Liu et al. 2003). The individual recombineering steps for the generation of the targeting vector are presented schematically in Fig. 3.20, and the strategy for the generation of the Munc13-1 CKO mouse line is illustrated in Fig.3.21 A.

3.2.1.1. Retrieving of BAC DNA fragment into the PL253 vector

In the first step, a 9.7 kb fragment containing the targeted exon 21 and the flanking long- and short arm for homologous recombination in ES cells was retrieved via recombineering from the BAC into the pBluescript-based targeting vector backbone PL253, containing a modified MC1TK cassette (Liu et al. 2003) (Fig. 3.20). First, the BAC clone bMQ 441l13

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and the bacteria were plated on LB agar plates containing chloramphenicol to select for the presence of the BAC plasmid. Colonies were chosen and BAC DNA was purified. 10 μl of purified BAC DNA before and after transformation was digested overnight at 37°C with 20 units of the restriction enzymes SalI, NruI and ClaI in 50 μl volumes each. The respective restriction patterns were compared after pulsed field gel electrophoresis on an agarose gel. The expected band pattern for all restriction enzymes was found in all clones tested, with the exception of NruI, which did not cut at one predicted restriction site (data not shown). This finding indicated small differences between the C57BL/6J reference sequence and the SV129 ES cell DNA. Indeed, during the generation of the Munc13-1 CKO targeting vector, some minor differences to the C57BL/6J reference sequence were observed after sequencing. However, these differences in the nucleotide sequence did not alter the identity of the encoded amino acids and were therefore considered to reflect genomic polymorphisms between strains.

In the next step, short homology arms (labeled A and B; Fig 3.20.1) were amplified from the BAC clone using PCR and subcloned into PL253, resulting in the plasmid PL253-AB (see 2.2.2.16.1.). These short homology arms define the borders of the desired sequence to be retrieved from the BAC clone. For the retrieval of the 9.7 kb BAC fragment into the PL253 retrieval vector, 600 ng of the PL253-AB DNA was linearized by 20 units HindIII for 2 h. Next, 170 ng DNA was electroporated into electrocompetent EL350 cells that contain the bMQ 441l13 BAC and in which the expression of the gap repair enzymes was induced. Amp^r colonies (i.e. colonies transformed with PL253-AB) were screened by PCR for the integration of the 9.7 kb Munc13-1 genomic sequence into the PL253-AB retrieval vector. In three independent experiments, 120 colonies were screened but gave rise to only one positive clone. To select against plasmid multimers, which can occur in pBluescript-based vectors by rolling circle replication during the gap repair process, 1 ng of the gap-repaired plasmid was retransformed into Electro10 Blue cells and Amp^r colonies were selected and tested for the presence of the targeted plasmid.

3.2.1.2. Targeting of the first loxP site upstream of Munc13-1 exon 21

In order to introduce one loxP site upstream exon 21 of the Munc13-1 gene, a Neo cassette flanked by two loxP sites was first introduced into the Munc13-1 genomic sequence via site-specific recombineering (Fig. 3.20.2). In a subsequent step, the Neo cassette was excised by mediated recombination after L-arabinose induced Cre-recombinase expression in SW106 E.coli cells. The Neo resistance gene that was used

(from the PL452 vector) is flanked by two loxP sites and is regulated by two promotors:

the phosphoglycerate kinase I (PGK) promotor, which permits the expression of the Neo resistance gene in eukaryotic cells, and the EM7 promotor, which enables prokaryotic cell expression. Moreover, the Neo cassette is followed by a bovine growth hormone polyadenylation site (bpA), a termination signal for protein expression in eukaryotic cells.

Short homology arms (Labeled C and D; Fig. 3.20.2) were PCR amplified from the BAC DNA and subcloned into PL452 (See 2.2.2.16.2.). The Neo cassette flanked by the two short homology arms was cut out of the resulting PL452 vector by digestion with 20 units of KpnI and NotI as well as ScaI (to cut the plasmid backbone in the ampicilin resistance gene). 100 ng of the C-loxP-PGK-EM7-NeobpA-loxP-D fragment were electroporated together with 10 ng of the PL253 vector containing the 9.7 kb Munc13-1 genomic insert into electrocompetent and induced SW106 cells and Amp^r (PL253 vector) and Kan^r (Neo cassette) colonies were selected. Five independent experiments were performed, in total

~450 clones were screened, however only the last attempt gave rise to 11 positive colonies. Positive colonies contained a mix of gap-repaired and non-gap-repaired plasmids. Therefore, 1 ng of the gap-repaired plasmid was retransformed into XL1-Blue cells and colonies resistant to Amp^r and Kan^r were selected. 1 ng of the retrieval vector containing the loxP-PGK-EM7-NeobpA-loxP cassette was then electroporated into electrocompetent SW106 cells after arabinose-induced Cre-expression. Cre activity resulted in the removal of the Neo cassette by Cre-mediated recombination between the two loxP sites in all selected Amp^r colonies, leaving only one loxP site upstream of Munc13-1 exon 21.

3.2.1.3. Targeting of the second loxP downstream of Munc13-1 exon 21

In order to introduce the second loxP site and a Neo cassette for positive selection in ES cells, a Neo cassette, flanked by two flippase (FLP) recombinase target (FRT) sequences and followed by a single loxP site was integrated downstream of Munc13-1 exon 21 by site-specific recombination in E.coli cells. Generation and subcloning of short homology arms amplified from the BAC DNA (termed E and F: Fig. 3.20.3) into PL451 is described in 2.2.2.16.3. The expression of the Neo resistance gene is controlled by a PGK and an EM7 promotor together with a bpA site. The Neo cassette, flanked by the two short homology arms, was cut out of the E-FRT-PGK-EM7-NeobpA-FRT-loxP-F containing

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NeobpA-FRT-loxP-F fragment were electroporated together with 10 ng of the PL253 retrieval vector from the previous step into electrocompetent and induced cells. Amp^r (PL253 vector) and Kan^r (Neo cassette) colonies were selected. 32 colonies were screened and four positive clones were obtained, which contained a mixture of gap-repaired and non-gap-gap-repaired plasmids. 1 ng of the plasmid DNA was retransformed into XL1-Blue cells and Amp^r and Kan^r colonies were selected. Positive clones contained a single FRT-PGK-EM7-NeobpA-FRT-loxP cassette downstream of the Munc13-1 exon 21 in addition to the one loxP site upstream of exon 21 (Fig. 3.23.3 and 3.24 A).