Conditional targeting of the TβRII gene.

(A) The wild-type TβRII locus was targeted by homologous recombination with a gene construct containing insertions of theneo gene flanked by loxP sites (arrowheads) upstream of exon 4, a single loxP site downstream of exon 4, and the HSV-tk gene at the 3′ flank of the construct. Homologous recombinants were identified using the PCR primers P1 (external) and P2 whereas the primers P6 and P7 verified retention of the single loxP site. Transient Cre-expression in targeted ES cells generated clones with a “floxed” or a null allele, respectively. PCR for screening of “floxed” and null mutants following Cre/lox-recombination in ES cells was done by using the P1 and P2 primers to verify excision of neo and the P6 and P7 primers to determine the presence or absence of exon 4. The primer pairs P3/P4 and P3/P5 were used to screen for germline transmission of the “floxed” and the null alleles, respectively. (B) PCR screening of neo-resistant clones for homologous recombination using P1 and P2 to amplify a 3.6-kbp recombinant sequence. M indicates 1 kbp molecular weight marker. (C) Germline transmission of the “floxed” allele in samples 1 and 2 as shown by the presence of a 575-bp PCR product amplified from tail DNA by the P3 and P4 primers. R1 indicates TβRII+/flox ES-cell DNA; R2, TβRII^+/− ES-cell DNA; R3, wild-type DNA.