Fig. 1. Disruption of the Tbx4 gene by homologous recombination in ES cells. (A) 7.3 kb of mouse genomic DNA was used to create a targeting construct containing a loxP site inserted into intron 4, a floxed PGK-neo PGK-thymidine kinase dual selection cassette (neo tk) inserted into intron 5 and a diptheria toxin (DT) gene 5' of the homologous DNA for negative selection against random integration. The indicated region of the Tbx4 cDNA was used for in situ hybridization (in situ probe). E, EagI; X, XhoI; Xb, XbaI; K, KpnI; S, SpeI; RV, EcoRV. Solid arrowheads indicate loxP sites. Boxes indicate exons: black, untranslated regions; white, coding regions; gray, T-box domain. Labeled boxes indicate selection cassettes. Figure not drawn to scale. (B) After electroporation, G418-resistant colonies were screened for homologous recombination by Southern hybridization of EagI/XbaI genomic digests probed with the 3' external probe shown in A. (C) Homologous recombination of the 5' end of the vector, including the loxP site, was confirmed using a KpnI digest probed with the 5' internal probe shown in A. (D) Oligonucleotides a and b were used to amplify the indicated genomic region by PCR, producing a 500 bp endogenous band and a slightly larger Tbx4^tm1Pa band with the loxP site insertion. Recombination of the Tbx4^tm1Pa allele was achieved by mating to a cre-expressing mouse. The recombined Tbx4^tm1.1Pa allele was genotyped using oligonucleotides a, b and c, to produce the same 500 bp endogenous band and a 260 bp mutant band.

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