Fig. 3.

Taf1 1loxP hemizygous male embryos developed to blastocysts but resulted in no embryonic stem cells. (A-F) Timed matings between 1loxP heterozygous females from Fig. 2B and Wt males (A-E) or between 2loxP heterozygous females from Fig. 2A and Wt males (F). (A,B) Harvest was at embryonic day (E) 9.5. (A) Dissection of decidua resulted in 15 empty decidua and 15 containing embryos within yolk sacs. Statistical analysis showed that the empty:embryo ratio was not significantly different from that expected assuming that empty decidua were induced by subsequently lost 1loxP embryos. Genomic DNA amplification of embryo-derived tissues resulted in detection of no 1loxP embryos of either sex. Statistical analysis showed that the female:male ratio was not significantly different from that expected. However, regardless of sex, or considering males only, the loxP:Wt ratio was highly significantly different from expected, as 0% of embryos carried a recombined 1loxP. (B) Representative images of empty and embryo-containing decidua from A. Arrowhead, yolk sac; arrow, embryo. Scale bar: 2 mm. (C-F) Harvest was at E3.5. (C) Blastocysts were harvested from both the B6 and B6129F1 mice. Surprisingly, genomic DNA amplification detected 1loxP embryos of both sexes from both B6 and B6129F1 mice. Statistical analysis of the total blastocysts showed that both the female:male and loxP:Wt ratios were not significantly different from those expected, and 49.1% of blastocysts carried a recombined 1loxP. (D) Representative genomic DNA amplification results for the four genotypes recovered as blastocysts in C. Primer locations are indicated in Fig. 1B and sequences in Table S2. L, 100 bp ladder; +, positive control ear notch from a 1loxP heterozygous female; -, negative control ear notch from a B6-Wt male; NT, no template; F, female; Het, heterozygote; Wt, wild type; M, male; Hemi, hemizygote; recomb., recombined loxP sites. Note: all blastocysts were also genotyped and found to be negative for 2loxP. L, 100 bp DNA ladder; NT, no template. (E) Blastocysts were harvested from B6129F1 mice and cultured to establish embryonic stem cells (ESCs). Genomic DNA amplification of ESCs resulted in detection of 1loxP heterozygous female cell lines, but no 1loxP hemizygous male cell lines. Statistical analysis showed that the female:male ratio was not significantly different from that expected but did show a trend towards less male cell lines. However, regardless of sex, or considering males only, the loxP:Wt ratios were significantly different from those expected, as only 15.8% of embryos carried a recombined 1loxP. (F) Blastocysts were harvested from B6129F1 mice and cultured to establish ESCs. Genomic DNA amplification of ESCs resulted in detection of 2loxP embryos of both sexes. Statistical analysis showed that the female:male ratio was not significantly different from that expected. Furthermore, regardless of sex, or considering males only, the loxP:Wt ratios were not significantly different from those expected, as 63% of cell lines carried a recombined 1loxP. Statistical analyses used the two-tailed binomial test for sex ratios and one-tailed binomial test for loxP:Wt as only loss of loxP embryos or cell lines was anticipated. Red, females; blue, males.