Figure 1.
Figure 1. Targeting strategy and confirmation of the recombined SCL genomic locus. (A) Schematic overview of the targeting strategy. In the upper representation the SCL wild-type genomic locus is shown. Coding exons (IV, V, and VI) are depicted as black, and noncoding exons (Ia, Ib, IIb, III, and part of VI) are depicted as white boxes. The targeting construct is shown below the SCL genomic locus, consisting of 2 homology arms, the tTA-2S coding sequence (striped box), and the floxed neomycin-resistant selection cassette (gray box). In the targeting construct all ATG codons in exon IV and the first ATG in exon V were changed to GGG codons. LoxP Cre-recombinase recognition sites flanking the neomycin cassette are indicated as black triangles. Below the targeting construct the recombined mutant SCL locus is shown still containing the neomycin cassette (Neo+). At the bottom of the representation the recombined SCL locus is depicted after excision of the neomycin cassette (Neo-). H indicates Hind III; R, EcoRI; N, Not I; X, XbaI;A, ApaI, and B, BamHI. (B) 5′ confirmation of the recombined SCL locus by Southern blotting using a specific outside probe. Digestion with Hind III of wild-type (WT) DNA gives rise to a 13-kb fragment, whereas the correctly recombined locus will result in a smaller 11.2-kb fragment (GT1 and GT2, germ-line-transmitting mouse founder line 1 and 2). (C) 3′ confirmation of the recombined SCL locus by Southern blotting. BamHI digestion of genomic DNA followed by hybridization with an inside probe produces a 4.9-kb fragment for the wild-type allele (WT) and a 2.4-kb fragment for the mutant knock-in allele (GT1). (D) In vivo excision of the neomycin-resistant cassette. PCR was used to verify the excision of the neomycin-resistant cassette from the germ-line of the SCL tTA-2S knock-in mouse. The recombined SCL locus still containing the cassette will produce a 1491-bp amplification product (Neo+). After excision of the neomycin cassette the same primers will amplify a 242-bp fragment (Neo-). The 764-bp amplification product is specific for the SCL wild-type allele.

Targeting strategy and confirmation of the recombined SCL genomic locus. (A) Schematic overview of the targeting strategy. In the upper representation the SCL wild-type genomic locus is shown. Coding exons (IV, V, and VI) are depicted as black, and noncoding exons (Ia, Ib, IIb, III, and part of VI) are depicted as white boxes. The targeting construct is shown below the SCL genomic locus, consisting of 2 homology arms, the tTA-2S coding sequence (striped box), and the floxed neomycin-resistant selection cassette (gray box). In the targeting construct all ATG codons in exon IV and the first ATG in exon V were changed to GGG codons. LoxP Cre-recombinase recognition sites flanking the neomycin cassette are indicated as black triangles. Below the targeting construct the recombined mutant SCL locus is shown still containing the neomycin cassette (Neo+). At the bottom of the representation the recombined SCL locus is depicted after excision of the neomycin cassette (Neo-). H indicates Hind III; R, EcoRI; N, Not I; X, XbaI;A, ApaI, and B, BamHI. (B) 5′ confirmation of the recombined SCL locus by Southern blotting using a specific outside probe. Digestion with Hind III of wild-type (WT) DNA gives rise to a 13-kb fragment, whereas the correctly recombined locus will result in a smaller 11.2-kb fragment (GT1 and GT2, germ-line-transmitting mouse founder line 1 and 2). (C) 3′ confirmation of the recombined SCL locus by Southern blotting. BamHI digestion of genomic DNA followed by hybridization with an inside probe produces a 4.9-kb fragment for the wild-type allele (WT) and a 2.4-kb fragment for the mutant knock-in allele (GT1). (D) In vivo excision of the neomycin-resistant cassette. PCR was used to verify the excision of the neomycin-resistant cassette from the germ-line of the SCL tTA-2S knock-in mouse. The recombined SCL locus still containing the cassette will produce a 1491-bp amplification product (Neo+). After excision of the neomycin cassette the same primers will amplify a 242-bp fragment (Neo-). The 764-bp amplification product is specific for the SCL wild-type allele.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal