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Figure 7


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 Tbx4floxNeoHLEA, a floxed HLEA knock-in allele, and Tbx4{Delta}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 Tbx4floxNeoHLEA band of 9.1 kb with an external 3' probe (see A). (C) Mice carrying the Tbx4floxNeoHLEA allele were crossed to a Cre deleter strain to generate the Tbx4{Delta}HLEA allele. Genotyping by PCR confirmed the presence of heterozygous and homozygous animals. (D) The relative expression levels of wild-type 129P2 and {Delta}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 {Delta}HLEA 129P2 to DBA. (E-J) Whole-mount in situ hybridization for Tbx4 mRNA in wild-type and homozygous {Delta}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.





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