G ENE TARGETING AND BREEDING STRATEGIES

4.1.1 Knockout-first-allele targeting strategy

For the generation of all mouse models during this work, the EUCOMM/KOMP (European conditional mouse mutagenesis program/ knockout mouse project) “knockout-first allele” (KO-1st) targeting approach was used (Skarnes, Rosen et al. 2011). For both katanin and spastin, the targeting strategies relied on the identification of one or more critical exons that were common to all known transcript variants and which, when deleted, created a frameshift mutation.

In addition to the loxP-flanked exons determined for the excision with Cre recombinase and the two homology arms, this KO-first allele contained a gene-trapping cassette flanked by a FRT site at the 5’ end and by a loxP site at the 3’ end followed by a neomycin selection cassette which was flanked by an additional FRT site at its 3’ end.

The gene-trapping cassette (Figure 18) consisted of a promoterless LacZ gene that was flanked by an upstream 3’ En2 SA (engrailed-2 splice acceptor) site followed by an IRES (internal ribosomal entry site) sequence and a downstream transcriptional termination sequence (polyadenylation sequence; polyA).

Figure 18: Example of a promoter-driven KO-first-allele targeting vector. Shown is an exon (black rectangle) to be targeted characteristically flanked by LacZ (β galactosidase cDNA), neo (neomycin phosphotransferase cDNA), 5’ and 3’ homology arms (5’ ha and 3’ ha), FRT recognition sites (green semicircles) and loxP recognition sites (grey triangles). En2SA: engrailed-2 splice acceptor; IRES: internal ribosomal entry site; pA: polyadenylation signal; hbactP: human β actin promoter; AmpR: ampicillin resistance; BsdR: blasticidin S deaminase resistance;

PGK: phosphoglycerate kinase promoter; DTA: diphtheria toxin A.

This targeting strategy was chosen for reporting the endogenous promoter activity of the targeted gene because the gene-trapping cassette is transcribed from the endogenous promoter in the form of a fusion transcript, in which the exons upstream of the insertion site are spliced in frame to the reporter gene. The transcription is terminated prematurely at the polyadenylation site, and the processed fusion transcript encodes a truncated and non-functional version of the cellular protein and the reporter.

An autonomous hbactP (human β actin promoter) driven neomycin selection cassette allowed to enrich successfully recombined ESC clones after electroporation with the targeting vector. The

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selection cassette could be removed via FRT sites or via loxP sites depending on the downstream experiments. Additionally, the plasmid backbone contained a PGK (phosphoglycerate kinase) promoter-driven DTA (diphtheria toxin A) cassette for negative selection and AmpR and BsdR for plasmid amplification before gene targeting.

4.1.2 Breeding strategy for katanin and spastin knockout mouse lines

The KO-first-allele can be used for multiple purposes and can produce reporter KOs, conditional KOs, and null-alleles depending on the sequence of exposure to the site-specific recombinases Flp and Cre (Figure 18). This required sequential breedings to different transgenic Cre and Flp driver lines. The embryonic stem cells for gene targeting of both katanin and spastin were derived from C57BL/6N mice (Pettitt, Liang et al. 2009) and the KO-1st animals obtained in filial generation 1 (F1) were backcrossed to the C57BL/6J genetic background in 5 filial generations.

The breedings using Cre and FLP driver lines with following outcrossing of these transgenes further added to the backcrossing. Following the breeding scheme developed in Table 19, conditional KO mice were generated with an F5 backcrossed C57BL/6J background.

The C57BL/6N ES cells used for the generation of chimeric founder mice had an agouti locus leading to a brown fur in their offspring. Thus, brown chimeric animals were selected for the generation of F1 lines.

Table 19: Overview of the sequential breedings to generate mice containing KO-first (KO 1-st), floxed (FL), conditional KO, null (KO) and reporter alleles (GT) for the genes of interest (GOI, i. e. spastin or katanin) starting with chimeric mice in the F0 generation. F0-F5: filial generations with one additional breeding using a mouse line in the BL57/6J genetic background. For better orientation, the individual alleles are highlighted in different colors.

F0 GOI^{WT/KO 1-st} chimera (C57BL/6N background)

F1 GOI^WT/KO1-st KO 1-ST ALLELES

F2 GOI^WT/FL/FLP^WT/TG (after FLP mating) FLOXED ALLELES

GOI^WT/GT/Cre^WT/TG F3 GOI^WT/FL/FLP^WT/WT (after crossing out FLP) GOI^WT/GT/Cre^WT/WT F4 GOI^WT/FL/CreWT/CamKII-TG GOI^WT/FL/Cre^WT/WT GOI^WT/KO/Cre^WT/TG

F5

GOI^WT/WT/CreWT/CamKII-TG, GOI^WT/WT/Cre^WT/WT GOI^WT/FL/CreWT/CamKII-TG, GOI^WT/FL/Cre^WT/WT

GOI^FL/FL/CreWT/CamKII-TG, GOI^FL/FL/Cre^WT/WT

GOI^WT/KO/Cre^WT/WT GOI^KO/KO/Cre^WT/WT

CONDITIONAL KO ALLELES NULL-ALLELES REPORTER KOs

4.1.3 Forebrain-specific Cre-driver lines

In order to generate postnatal and forebrain-specific KOs for spastin and katanin, CamKIIα-Cre transgenic mice were used. Several transgenic CamKIIα-Cre-driver lines were tested by using a ROSA26 knock-in reporter strain developed by Soriano et al. in order to test the expression and activity of Cre recombinase (Figure 19a). In this reporter strain, two loxP sites flank a STOP

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64 codon upstream of a LacZ reporter cassette, preventing its expression in the absence of Cre-mediated recombination. Following Cre-Cre-mediated recombination, the stop codon is deleted and allows the expression of the reporter. LacZ staining of brains from offspring resulting from CamKIIα-Cre and ROSA26 Cre reporter strain breedings at different developmental stages were done. For the transgenes originating from the transgenic lines developed by Casanova et al. and Minichiello et al., prenatal expression of CamKIIα-Cre was already observed at different extent (Figure 19c). In contrast, the transgene reported by Tsien et al. showed no apparent LacZ activity until postnatal day 2, with a starting expression of Cre at postnatal day 8 and a full forebrain expression in adult mice (Figure 19c and b). In the cerebellum, no LacZ activity was observed as well as in the negative controls only containing the R26R reporter locus (data not shown).

Figure 19: LacZ staining of tissues from offspring resulting from different CamKIIα-Cre-driver lines (Casanova, Minichiello, Tsien) mated to the R26R reporter line (Soriano et al. 1999) to visualize Cre-mediated recombination. a) The CamKIIα-Cre driver lines to be tested were bred to the ROSA26 Cre reporter line. b) Expression pattern of β-galactosidase in adult mouse brain resulting from a mating using the CamKIIα-Cre driver line developed by Tsien et al.. c) Expression of β-galactosidase in prenatal mouse brains using CamKIIα-Cre driver lines originating from Casanova et al. (E18) and Minichiello et al. (E15). d) β-galactosidase expression in brains isolated from offspring resulting from matings using the CamKIIα-Cre driver line from Tsien et al. (E16, P2, P8, P14 and P21).

To rule out the possibility of occasional germline expression of Cre in conditional KO mouse lines, a PCR method was developed to control for this event using two primers outside and one primer inside the floxed region (Figure 20, bottom left). However, as can be concluded from the electropherogram in Figure 20, a band corresponding to the size of a null-allele could occasionally be observed using template-DNA extracted from tail-tip biopsies in theoretically forebrain-specific conditional-KO mouse lines containing the CamKIIα-Cre transgene from the Tsien et al. origin (see lanes 2 and 4, Figure 20).

a) b)

c) Casanova Minichiello d) Tsien

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Figure 20: PCR method to control for CamKIIα-Cre expression in germ cells. Genotyping PCR using tail-tip DNA extracts from offspring resulting from matings of GOI^WT/FL females (mouse shown in red) with GOI^FL/FL/CamKII-Cre^WT/TG males (mouse shown in blue). As shown by agarose gel electrophoresis, besides the expected band sizes for GOI^WT/FL and GOI^FL/FL mice also bands at the size of GOI^WT/KO mice (with a null-allele, lanes 2 and 4) were obtained. The results were obtained using samples from one litter. GOI: gene of interest. M:

molecular weight marker.

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