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Figure 4


Fig. 4. Targeted alleles of Nkx2-5. (A) Targeting strategies used to create Nkx2-5 `knock-in' alleles (see text). (B,C) Wild-type Nkx2-5 locus with targeting vectors and targeted alleles shown before and after Cre deletion of drug selection cassettes (H, pgkHygro; N, pgkNeo). Coding region exons shaded black. Arrows show loxP sites. {Delta}HD indicates the small homeobox deletion (amino acids 141-184). 5'P and 3'P, and p1-p4, respectively, indicate positions of Southern probes and PCR primers for validation of recombinations and deletions. Restriction endonucleases: Nc, NcoI; Bc, BclI; Bg, BglII; M, MluI; N, NotI; X, XbaI; Xh, XhoI. (D) Southern blot analysis of ES cell DNA of indicated genotype digested with BclI (left) or NcoI (right) using probe 3'P. The 23 kb band is the wild-type allele, while 9 and 6 kb bands represent targeted alleles. (E) PCR validation of Cre-deleted ES cell clones using primers specific for the pgkHygro cassette, as indicated in B and C, and those specific for the GFP and pgkNeo cassettes.