Figure 1.
Structure and expression of the Gtf2iΔex2 mutant allele. A. Schematic genomic diagram of the targeting strategy. a. Partial restriction map of wild-type genomic DNA encompassing exons 1 (grey box), 2, 3, 4 and 5 of the Gtf2i locus (filled boxes) (B, BamHI; E, EcoRI; Bg, BglII); the translation initiator sites in the wild type (*) and targeted (**) alleles are indicated. The arrows indicate the location of the primers used for RT-PCR analysis. The targeting vector contains the PGK-neo (open box, neo) and lox511 sequences (filled triangle). Predicted targeting allele after homologous recombination. The stippled box indicates the location of the probe used for Southern blot analysis, recognizing EcoRI DNA fragments of 14,7kbp (wild-type allele) and 9,7kbp (targeted allele). B. RT-PCR analysis. Using primers located in exons 1 and 4 (1) or 2 and 4 (2) of Gtf2i on mRNA from Gtf2i+/+, Gtf2i+/Δex2 and Gtf2iΔex2/Δex2 mice. Transcripts lacking exon 2 are observed in heterozygous and homozygous mutant mice (1), while no amplicon was obtained with exon 2 and 4 primers in Gtf2iΔex2/Δex2 mice (2). C. Western blot analysis. Total protein extracts from MEFs (Gtf2i+/+ ; Gtf2i+/Δex2 and Gtf2iΔex2/Δex2) and COS7 cells transfected with a Gtf2iΔex2 cDNA lacking the first four exons were used for western blot analysis. We can observed that the truncated form present in Gtf2i+/Δex2 and Gtf2iΔex2/Δex2 extracts co-migrate with the artificial truncated form expressed in COS7 cells. D. Subcellular localization of endogenous TFII-I in MEFs. The genotype of the cells is shown in the bottom. Equal nuclear localization of Δ140TFII-I and wild-type proteins was confirmed by DAPI staining in all cases. Scale bar: 10 μm.