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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.
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.
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