Fig. 2. The mRNA and protein products of the wild-type and the mutant C3G alleles. (A) Northern analysis of total RNA isolated from E10.5 embryos of C3G^gt/+ heterozygous intercrosses. 10 µg of total RNA were loaded per lane. The genotype of the embryos is indicated above, as wild type (wt), heterozygous mutant (ht) and homozygous mutant (mt) for the gene trap mutation in the C3G locus. (Upper panel) Hybridisation with the C3G probe 2 as indicated in Fig. 1A. (Middle panel) hybridisation with a lacZ-specific probe. (Lower panel) ethidium bromide staining of the 18S rRNA. Note the absence of detectable C3G mRNA in homozygous mutant embryos by this method. The lacZ probe detected a fusion mRNA of the expected size of 4.7 kb (169 bases 5' UTR and coding sequence 5' of the gene trap insertion of the C3G locus and 4.42 kb mRNA product of pGT1.8geo plus polyadenylation tail). (B) RT-PCR of total RNA isolated from macroscopically normal E10.5 embryos of heterozygous intercrosses. Oligo-dTTP was used to generate cDNA. Lanes 1, 2, 3, 4 and 5: cDNA of wild-type embryos used diluted 1:10 (1), 1:50 (2), 1:100 (3), 1:500 (4), 1:1000 (5). Lanes 6, 7, 8, 9 and 10: cDNA of homozygous C3G^gt/gt embryo was used diluted 1:10 (6), 1:50 (7), 1:100 (8), 1:500 (9) 1:1000 (10). Lane 11: control without cDNA template. M=100 bp ladder, intense band=500 bp. Note that even the 1:1000 diluted wild-type RNA yielded a prominent RT-PCR product, whereas the amount of product yielded from 1:10 and 1:50 dilution of the homozygous RNA was small. (C) PCR of undiluted homozygous mutant (mt) and wild-type cDNA (wt) as in B, but using primers spanning exons 1-21 representing all coding exons of the C3G locus. Note the presence of a small amount of product from homozygous template indicating the generation of normal protein coding mRNA from the homozygous mutant allele. Lanes 1 and 3 are controls. M=1 kb ladder; intense band=5 kb. (D) Western analysis of cell lysates of primary embryonic fibroblasts isolated from E10.5 homozygous, heterozygous and wild-type littermates. 40 µg of lysate were loaded per lane. Genotypes are wild type (wt), homozygous mutant (mt) and heterozygous (ht). C3G protein bands were detected with an anti-C3G antibody in the expected position (compare to Posern et al., 2000). Note the two prominent C3G bands in wild-type and heterozygous cell lysates. The two weak bands visible in homozygous lysate are likely to be residual C3G protein. They comprise no more than 5% of the normal level of C3G protein as determined by densitometry.