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. 2011 Jan-Feb;1(1):49–59. doi: 10.4161/bioa.1.1.15172

Figure 4.

Figure 4

Design and screening of −9ab ES cells. (A) The γTm gene showing the subcloned 6.7 kb SpeI fragment containing exons 9a and 9b with flanking intron sequence. The targeting construct with a LoxP sequence and a cassette containing LoxP—pGK Thymidine Kinase—pGK NeomycinR—LoxP. A 1.2 kb probe located outside the construct region is shown. (B) Southern blot analysis of C57BL/6 ES cell DNA following EcoRV digestion shows the expected 5.5 kb band for the WT allele and a 3.1 kb band for the recombined allele (Recomb) following probing with the 1.2 kb probe. (C) Following Cre recombinase and Gancyclovir selection the resulting alleles expected are WT, a floxed exon 9a + 9b or a full 9ab KO allele. Oligonucleotide primer sets common to the WT and both altered alleles are shown. (D) Genomic DNA isolated from ES cell clones was screened by PCR analysis. WT: +/+, Parental: homologously recombined clone containing exons 9a + b and the TKNEO cassette, −9ab+/−: clone lacking exons 9a + 9b and −9abFlox: clone containing a floxed exon 9a + 9b allele. PCR analysis outcomes from the primer set 9723F and 9b3′R is shown in the upper part and primer set 9724F and 9b3′R lower part. (E) Following re-targeting of the −9ab+/− clone with the −9ab construct the expected outcomes are a 9ab KO and homologously recombined alleles. (F) Genomic DNA isolated from ES cell clones was screened by PCR analysis using primer sets 9724F and 9b3−R to yield a 400 bp band and 9723F and 9b3′R to yield a 315 bp band. (G) Following the second round of Cre recombination and selection the expected outcome will be two −9ab−/− alleles. An oligonucleotide primer set common to both WT allele and the fully deleted exon 9a + 9b allele was used in order to confirm any −9ab−/− ES cell clones. (H) Genomic DNA isolated from ES cell clones were screened by PCR analysis. A single −9ab−/− ES cell clone was determined by long range PCR against both WT+/+ and −9ab+/− controls. The expected 2.6 kb fragment for the WT allele and a 400 bp fragment for the deleted exons 9a and 9b is shown.