Figure 1.

Generation of a transgenic Ink4a/Arf allele. (A) Structure of the transgene. The transgene contains the complete exon structure of three genes, Ink4b, Arf, and Ink4a. Boxes labeled SP6 and T7 represent short terminal sequences (of 20 bp) used for the identification of the transgene by PCR (see Materials and Methods). A thick bar near the SP6 terminus indicates the location of the probe used for the Southern blot shown in C. (B) Fluorescent in situ hybridization of the transgene in a representative MEF metaphase. The fluorescent probe consists of the complete transgene. The transgene is located in a chromosome that is smaller than the two homologs containing the endogenous alleles. (C) Southern blot to detect the SP6-terminal region of the transgene. Genomic DNA digested with BamHI produced a band of ∼4 kb, present in both wild-type and transgenic mice, and a single additional band of ∼6 kb specific to transgenic mice. The probe used consisted of a PCR fragment of ∼380 bp (schematically shown in A) that was generated by primers SP6-PAC and SP6-tg (see Materials and Methods). (D) PCR genotyping of the various Ink4a/Arf alleles. (Top) Detection of the transgene by a PCR reaction based on the T7-terminal sequence of the transgene. (Bottom) The endogenous Ink4a/Arf allele was indirectly genotyped through a linked polymorphism in the Tyrp1 (tyrosinase-related protein 1) locus, which is located at a genetic distance of ∼4.7 cM. The Ink4a/Arf-wild-type (wt) allele is linked to the wild-type Tyrp1 allele, whereas the Ink4a/Arf-null allele is linked to the mutant Tyrp1(brown) allele (Sviderskaya et al. 2003). The two Tyrp1 alleles can be distinguished by a diagnostic TaqI site (Zdarsky et al. 1990) present in Tyrp1-wt (linked to Ink4a/Arf-wt), but absent in Tyrp1(brown) (linked to Ink4a/Arf-null). A PCR product that contains the reported TaqI polymorphism was digested with TaqI to determine the presence or absence of the TaqI site.