Analysis deltaEF1 protein function in vivo by gene targeting

为了阐明deltaEF1在胚胎发生中的可能作用，我们利用小鼠胚胎干细胞进行基因打靶来阻断deltaEF1基因。我们期待这些结果对胚胎发生的影响，这将有助于我们深入了解deltaEF1在活体中的功能。首先，我们利用鸡的cDNA通过与小鼠的同源杂交的方法克隆了小鼠的deltaEF1同源物。对克隆的小鼠基因组和cDNA进行了部分鉴定。我们发现DeltaEF1在两个物种之间是保守的，并且小鼠的DeltaEF1在N端和C端也有两个成簇的锌指区域，在其中间部分有同源结构域样序列。第二，我们构建了一个靶向载体，使得与DNA结合的C端锌指部分可以被截断，导致靶基因没有功能。以E14小鼠胚胎干细胞为靶向载体，进行电穿孔实验。我们获得了一个在两条同源染色体的其中一条染色体上存在deltaEF1基因突变等位基因的ES细胞克隆。最后，我们用靶向ES细胞注射的囊胚(来源于C57BL/6和C3H小鼠的F1)产生了小鼠嵌合体。其中一个嵌合体可以产生后代，其后代具有来自ES细胞的deltaEF1基因的中断等位基因。这些杂合子的动物看起来很正常，很能生育。为了了解deltaEF1蛋白在体内的功能，我们现在将通过杂合子小鼠的杂交获得纯合子动物，并观察其对胚胎发育的影响。

To clarify the possible role of deltaEF1 in embryogenesis, we undertook the disruption of deltaEF1 gene by gene targeting using mouse embryonic stem (ES) cells. We expect the affects of those results on embryogenesis which should give us insight into the function of deltaEF1 in vivo.First, we cloned the mouse deltaEF1 homologue using chicken cDNA by cross-hybridization to the mouse counterpart. The cloned mouse genomic and cDNAs were partially characterized. We found that the deltaEF1 is well conserved between the two species and that the mouse deltaEF1 has also two clustered zinc finger regions in N-terminal and C-terminal proximal region and the homeodomain-like sequence in its middle portion.Second, we constructed a targeting vector so that the C-terminal zinc finger portion, which has the essential function to bind to the DNA, can be truncated, resulting in making the targeted gene nonfunctional. Using E14 mouse embryonic stem ES cells, we performed electroporation with the targeting vector. We have obtained one ES cell clone which has a disrupted allele of deltaEF1 gene on one of the two homologous chromosomes.Finally, we produced mouse chimeras from blastocysts (derived from F1 of C57BL/6 and C3H mice) which were injected with the targeted ES cells. One of the chimeras could generate offspring which has the disrupted allele of deltaEF1 gene derived from the ES cells. These heterozygous animals look normal and fertile. In order to understand the in vivo-function of deltaEF1 protein, we are now going to get the homozygous animals by intercrossing the heterozygous mice and to see how the embryonic development can be affected.