Figure 3.

Design and screening of −9d ES cells. (A) The −9d^+/− homologously recombined (parental) allele containing the Neomycin cassette. An oligonucleotide primer set designed to the Neomycin cassette is also shown. (B) Following Cre-mediated recombination to remove the Neomycin cassette the expected −9d KO allele is shown with oligonucleotide primer sets common to both WT and −9d KO alleles. (C) Genomic DNA isolated from ES cell clones screened by PCR analysis. PCR analysis of outcomes from the three primer set F, R1 and R2 is shown in the upper part, with outcomes from the Neomycin primer set shown in the lower part. -ve, no template control; +/+, WT C57BL/6 ES cell DNA; −/−, genomic DNA from Δ9d deleted mouse line; Parental, homologously recombined −9d^+/− clone containing the NEO cassette; Clone#1, clone containing the NEO cassette; Clone#18, clone with NEO cassette removed. (D) Following re-targeting of the −9d^+/− ES clone (#18) with the −9d targeting construct, the possible outcomes from this re-targeting are WT (due to random integration), re-targeting back to the original 9d KO locus and targeting of the alternative allele. Oligonucleotide primer sets designed to detect the presence of exon 9d are shown. (E) Genomic DNA isolated from ES cell clones screened by PCR analysis using 9dF1 and 9dR1 primers. -ve, no template control; WT, C57BL/6 ES cell DNA; #1-#5 sample of the 330 clones. The predicted 527 bp fragment for exon 9d is shown. (F) The 330 ES cell clones were also analyzed by PCR for random and original locus re-targeting using F and R2 primers (upper part) and 9dF1 and 9dR1 primers (bottom part). -ve, no template control; WT, C57BL/6 ES cell DNA, −9d^+/− ES Clone#18; re-target, clone where −9d targeting construct has re-inserted into the original targeted locus; random, clone where −9d targeting construct has re-inserted randomly. The 900 bp fragment for knockout of exon 9d and the 527 bp fragment for presence of exon 9d is shown.