The genomic deletions were confirmed by internal genomic PCR (Fig. 1B, right panel). The structure of the resultant protein encoded by the Δex2 allele (Cas PI3K Inhibitor Library order Δex2) is shown at the bottom of Fig. 1A. As shown by western blotting (Fig. 1C), a protein of the expected molecular weight (∼90 kDa) was detected in the sample from CasΔex2/Δex2 mice (Cas Δex2), and this indicated correct targeting, the excision of Cas exon 2, and the production of the short Cas protein lacking the corresponding segment. Heterozygous (Cas+/Δex2) mice were apparently normal and were intercrossed to produce homozygous (CasΔex2/Δex2) mutants. No live CasΔex2/Δex2 neonates were obtained, and this indicated that the CasΔex2/Δex2

mice died as embryos. To investigate the date of embryonic lethality, embryos obtained from the intercrossing of Cas+/Δex2 mice were examined at different embryonic stages. As shown in Table 1, CasΔex2/Δex2 embryos were alive until

12.5 dpc, but no live CasΔex2/Δex2 embryos were obtained after 13.5 dpc. To examine the cause of embryonic lethality in CasΔex2/Δex2 mice, wild-type (WT) and CasΔex2/Δex2 embryos at approximately 12.5 dpc were subjected to pathological examination. A remarkable and progressive change was observed in the livers of CasΔex2/Δex2 embryos. At 11.5 dpc, when macroscopic change was not yet apparent (Fig. 2A, left panels), hematoxylin DMXAA cell line and eosin (HE)–stained sections revealed that part of the liver of a CasΔex2/Δex2 embryo began to collapse from the inside (Fig. 2B, second bottom panel). At 12.5 dpc, this change was more pronounced, and the liver capsule of a CasΔex2/Δex2 embryo was macroscopically 上海皓元 enlarged with a mass left inside (Fig. 2A, fourth panel). HE-stained sections showed that most of the hepatocytes were lost, whereas hematopoietic cells were preserved (Fig. 2B, fourth bottom panel). Despite intensive analysis, no pathological abnormalities were found in other organs, including

the brain, heart, lungs, spleen, and kidneys (data not shown). Therefore, we concluded that progressive liver degeneration was the primary cause of death of CasΔex2/Δex2 embryos. The pathological pictures suggested that the progressive hepatocyte reduction in CasΔex2/Δex2 embryos was due to apoptosis. To address this possibility, livers of CasΔex2/Δex2 and WT embryos 12.5 dpc were subjected to the TUNEL assay. As shown in Fig. 2C, no obvious staining was observed in the WT liver (left panels), whereas several round hepatocytes surrounding the central collapsed area were positively stained in the CasΔex2/Δex2 liver (indicated by arrowheads in the right bottom panel); this confirmed the apoptotic reduction of hepatocytes. To investigate the mechanism underlying hepatocyte apoptosis, localization of the Cas protein in the embryonic liver was examined by immunohistochemistry. Normal livers at 11.