Figure 1. Targeted disruption of Col5a3.

(A) Structure of the targeting vector and Col5a3 locus, before and after homologous recombination. Horizontal arrows mark directions of transcription of neo^r and tk cassettes. Blue, red, and hatched boxes represent COL1, C-propeptide, and 3′-UTR exons, respectively. The green boxes represent the Neo^r cassette; the yellow boxes represent 5′ and 3′ external probes. The asterisks mark the site of a premature stop codon engineered via blunt-end ligation of a NarI site. A, AflII; N, NarI. (B) Southern blot of AflII-restricted genomic DNA from wild-type and correctly targeted ES cell clones hybridized to the 5′ probe. (C) RT-PCR analysis of total RNA from 15.5-dpc embryos detected a 370-bp amplimer corresponding to wild-type Col5a3 RNA in wild-type (+/+) samples that was diminished in Col5a3^+/– (+/–) samples and absent in Col5a3^–/– (–/–) samples. Amplification of a GAPDH product was a loading control. (D) Immunoblotting of 15.5-dpc embryo homogenates detects pro-α3(V) chains in wild-type but not Col5a3^–/– samples. Reprobing with anti–β-actin antibody controlled for protein loading.