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Fig. 4 | BMC Genetics

Fig. 4

From: Generation of an 870 kb deletion encompassing the Skt/Etl4 locus by combination of inter- and intra-chromosomal recombination

Fig. 4

Skt/Etl4 gene inactivation strategies. a Overview of Skt/Etl4 wt (a) and mutant alleles (b-f) that were generated by gene targeting. (b) Integration of an IRES-lacZ-triple poly a into exon 1. Floxed PGK-Neo-pA was removed with Cre recombinase leaving a single loxP site 3’to lacZ. (c) LacZ-triple poly a integration into exon 5 with 3’loxP site after Cre removal of the floxed PGK-Neo cassette. (d) Cre-induced TAMERE between loxP sites of SktEx1IRESlacZ (b) and SktEx5lacZ (c) alleles in the mouse resulting in 595 kb genomic deletion between exon 1 and 5 (depicted in B). (e) Integration of floxed PGK-Neo cassette together with a GFP-tagged exon 21into the SktΔEx1-5 allele. (f) Cre mediated excision of 273 kb genomic sequences between integrated loxP sites in the SktΔEx1-5, Ex21GFP allele (depicted in B). Numbered boxes represent Skt/Etl4 exons, coding exons are marked in grey. lacZ-3pA: β-Galactosidase gene with triple poly a signal. Black triangle: lox P site. IRES: internal ribosome entry site. PGK-Neo-pA: Neomycin selection cassette with PGK promoter and poly a signal. GFP: green fluorescence protein tag with stop signal. PCR1 to PCR6 shows the positions of Primer pairs that were used for mouse genotyping shown in (c) and (d). b Scheme of the Skt/Etl4 locus drawn to scale showing the two deletion steps to eliminate Skt/Etl4 function. First TAMERE between the SktEx1IRESlacZ and SktEx5lacZ alleles generated a 595 kb deletion between exon 1 and 5. Second around 273 kb genomic DNA between exon 5 and 21 were deleted by Cre-mediated intrachromosomal excision. c PCR Genotyping of offspring after the first deletion event between exon 1 and 5 (see B) with Primer pair combinations PCR 1 to 4 depicted in a from breedings of wt males with SktEx1IRESlacZ/Ex5lacZ, ZP3::Cre females. Mice carrying the 595 kb deletion (SktΔEx1-5 allele, see A d) were positive for PCR 1 and 4 and negative for PCR 2 and 3. d PCR Genotyping of offspring’s after the second deletion event between exon 5 and exon 21 (see B) with PCR Primer pair combinations PCR1, 4, 5 and 6 depicted in a from breeding’s of wt males with SktΔEx1-5; Ex21GFP, ZP3::Cre females. Only when 273 kb of genomic DNA was removed positive signals with PCR 1 and 5 were obtained. In addition PCR 6 specifically detects the whole 870 kb deletion due to the flanking primer positions (red triangle). e Verification of chromosomal rearrangements by Southern blot analysis with genomic mouse DNA digested with PstI (a) or NcoI (b) and hybridized with a lacZ specific probe for discrimination of the different Skt/Etl4 alleles that were generated. A polymorphism obtained with EcoRV-digested genomic DNA (c) hybridized with a probe downstream of exon 21 (3‘exon21) probe was used to demonstrate the presence of the wt, heterozygous or homozygous SktΔEx1-20 alleles in mice. The corresponding restriction maps with the expected fragment sizes of Skt/Elt4 wt and mutant alleles are shown in Additional file 3: Figure S3. f Detection of endogenous SKT/ETL4 protein by Western blot analysis using anti-NGS antibody and protein lysates isolated from adult brain of wt and homozygous SktΔEx1-20 animals. Red arrowheads point to bands missing in lysates of mutant tissue

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