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

EAT/MCL1基因是在人胚胎癌细胞分化早期上调表达的基因，属于bcl2相关基因。我们先前已经证明，在培养的成纤维细胞中，它对化疗药物诱导的细胞凋亡具有抵抗作用，并且过表达该基因的小鼠出现胰岛增生。为了阐明EAT/MCL-1基因在体内的功能，我们打乱了胚胎干细胞中的EAT/MCL1基因，并产生了敲除小鼠。我们建立了11个胚胎干细胞系，其中EAT/MCL1外显子1的一个等位基因两侧带有loxP序列和一个选择盒。通过将目标细胞注射到小鼠早期胚胎中，产生了三个小鼠品系。与表达Cre的转基因小鼠杂交，我们产生了杂合子EAT缺失小鼠和牙线状小鼠。这种杂合子小鼠(Eat-/+)在形态上与野生型动物没有区别。这表明一半剂量的EAT/MCL-1足以满足正常发育。与组织特异性Cre表达的小鼠杂交，我们可以有条件地使EAT基因失效。我们引入了在上胚源性胚胎组织中表达cre的mox2cre转基因小鼠，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.