The function of the EAT, an inhibitor of apotptosis, in vivo and its molecular mechanism in disease

EAT/mcl 1基因是在人胚胎癌细胞分化早期表达上调的基因，属于bcl-2相关基因。我们以前已经证明，它赋予抵抗化疗药物诱导的细胞凋亡在培养的成纤维细胞和小鼠过表达这个基因表现出胰岛增生。为了阐明EAT/mcl-1基因在体内的功能，我们将EAT/mcl-1基因在胚胎干细胞中进行了敲除，建立了11个EAT/mcl-1基因外显子1的一个等位基因两侧带有loxP序列的胚胎干细胞系，并建立了一个筛选盒。通过将靶细胞注射到小鼠早期胚胎中产生三个品系的小鼠。与表达Cre的转基因小鼠杂交，我们产生了杂合EAT缺失小鼠和floxed小鼠。该杂合子小鼠（EAT-/+）与野生型动物在形态学上无法区分。这表明EAT/mcl-1的一半剂量足以正常发育。将floxed小鼠与组织特异性cre表达小鼠杂交，我们可以条件性地使EAT基因无效。我们已经引入了mox 2 cre转基因小鼠，其在外胚层衍生的胚胎组织中表达cre以敲除EAT基因。

EAT/mcl1 gene was isolated as a gene which was up-regulated at the early stage of differentiation of human embryonal carcinoma cells and belonged to the bcl-2 related gene. We have previously shown that it confered resistance to apoptosis induced by chemotherapeutic agents in cultured fibroblasts and mice overexpressing this gene exhibited hyperplasia of pancreatic islet. In order to clarify the function of the EAT/mcl-1 in vivo, we have disrupted the EAT/mcl1 gene in embryonic stem cells and generated knock out mice.We have established 11 embryonic stem cell lines in which one allele of the EAT/mcl1 exon 1 was flanked by loxP sequence and a selection casette. Three lines of mice were generated by injecting the targeted cells into murine early embryos. Crossing with Cre expressing transgenic mice, we have generated heterozygous EAT null mice and floxed mice. This heterozygous mice(EAT-/+) was morphologically indistinguishable with wild type animals. This indicates the half dose of EAT/mcl-1 is sufficient to normal development. Crossing the floxed mice with tissue specific cre expressing mice, we can nullify the EAT gene in conditional manner. We have introduced mox2 cre transgenic mice which express cre in epiblast derived embryonic tissues to knock out the EAT gene.