Transmission of knock-in alleles to mouse germ line. (A) Targeting strategy for introducing knock-in alleles, AML1-2 and AML1-3, into wild-type ES cells. Refer to the legend of Figure 2D for symbols. (B) Genotype of the germ line mice harboring the knock-in allele was determined by Southern blot analysis using XbaI-digested genomic DNA. The mutated allele was detected as a 7-kb fragment that hybridizes with the 3′ outside probe whereas the wild-type allele yields a band of 14 kb. Representative results are shown. WT indicates wild type; Homo, homozygous; and Hetero, heterozygous. (C) Semiquantitative RT-PCR analysis was used to evaluate the expression level of the knock-in genes in comparison with that of a housekeeping gene, HPRT. Results are shown for 2 wild-type and 2 homozygous mice that were littermates for each of the mutations (+/+, wild-type). Serially diluted cDNA pools were analyzed (see “Materials and methods”). Lanes a, b, c, and d indicate 5^-1, 5^-2, 5^-3, and 5^-4 dilutions, respectively. M indicates size marker. Expression levels of the exogenous genes from the knock-in alleles in the homozygous animals were almost the same as those observed for the endogenous AML1 gene in wild-type littermates.