Fig. 7. Targeted knockout of HLEA reduces expression of Tbx4 in hindlimbs. (A) Schematic showing homologous targeting of the Tbx4 locus in mouse ES cells to create Tbx4^floxNeoHLEA, a floxed HLEA knock-in allele, and Tbx4^HLEA, where the floxed HLEA allele has been deleted using Cre recombinase. Gray bars, first and second exons of Tbx4; yellow ovals, HLEA; triangles, loxP sites; red arrows, genotyping primers; Neo, neomycin resistance cassette. (B) Southern blot of ES cell clones digested with MfeI shows a wild-type (Wt) band of 24 kb and a targeted Tbx4^floxNeoHLEA band of 9.1 kb with an external 3' probe (see A). (C) Mice carrying the Tbx4^floxNeoHLEA allele were crossed to a Cre deleter strain to generate the Tbx4^HLEA allele. Genotyping by PCR confirmed the presence of heterozygous and homozygous animals. (D) The relative expression levels of wild-type 129P2 and HLEA 129P2 Tbx4 alleles were compared with a wild-type DBA Tbx4 allele in E11.5 hindlimbs and lungs. Black bars, expression ratio of wild-type 129P2 to DBA; gray bars, ratio of HLEA 129P2 to DBA. (E-J) Whole-mount in situ hybridization for Tbx4 mRNA in wild-type and homozygous HLEA knockout embryos. Lateral views of E10.5 embryos (E,F) and dorsal views of E10.5 hindlimb buds (G,H) and E12.5 hindlimbs (I,J). Arrowheads indicate anterior side of hindlimb buds.