CreERT2-mediated inactivation of Ptch. (A) Part of the wild-type (wt) murine Ptch allele and the targeting vector, which upon homologous recombination generates Ptch^flox that harbors loxP sites (△) 5′ and 3′ of exons 8 and 9 as well as a frt-flanked (▲) neomycine resistance cassette in intron 9. Following tamoxifen treatment, Cre-mediated recombination generates the inactivated Ptch^del allele. The external probe used for Southern blot analysis of ES-cell transfectants and primers used for mouse genotyping and amplification of transcripts are indicated (see “Materials and methods” for details). XI indicates XhoI. (B) Using the external probe, Southern blot analysis of XhoI-digested DNA distinguishes wt Ptch and Ptch^flox, which give rise to a 15 948- and 8640-bp fragment, respectively. (C) Primer pairs indicated on the left and depicted in panel A identify genomic wild-type Ptch, Ptch^flox, and Ptch^del alleles by PCR performed on either mouse tail DNA (lanes 1-3) or DNA preparations from mouse embryos (lanes 4-5). The 1735-bp fragment amplified by the primer pair Neo-F/p1011R.2 identifies all floxed Ptch alleles. Loss of the 843-bp fragment amplified with primers p910F.4/Neo-R demonstrates successful Cre-mediated recombination. (Lane 1) Homozygous wild-type Ptch alleles, (lane 2) heterozygous Ptch^flox/+ alleles, (lane 3) homozygous Ptch^flox/flox alleles, (lane 4) heterozygous Ptch^del/+, and (lane 5) homozygous Ptch^del/del. (D) Recombination efficiencies in tPtch^−/− mice (top panel) and vehicle-treated Ptch^flox/floxERT2^−/− mice (middle panel) from the indicated organs were analyzed on cDNA-level using primer pair mPtc11/mPtc7R (for location, see panel A). Recombination efficiency in bone marrow (BM) is shown separately (bottom panel). The amplification of a 451-bp fragment indicates transcripts of the Ptch^del allele. The fragment marked by an asterisk results from a normal Ptch splice variant, in which exon 10 is missing.²⁴  Amplification of mGapd or β-actin served as control. cDNA obtained from day-12.5-old embryos (E12.5) served as positive control. li indicates liver; ki, kidney; sp, spleen; lu, lung; he, heart; sm, skeletal muscle; ce, cerebellum; br, brain; th, thymus; sk, skin; and ntc, no template control.