The Function of Nonmuscle Myosin Heavy Chains

该实验室以前的工作表明，使用同源重组将小鼠体内的一个非肌肉肌球蛋白重链(NMHC)替换为另一个是可能的。例如，我们用NMHC II-A替换NMHC II-B，导致NMHC II-A在内源性II-B启动子的控制下表达。目前，我们正在研究用GFP标记的人NMHC II-B取代小鼠NMHC II-A的效果。将GFP-NMHC II-B-SV40 Polya Casette导入NMHC II-A基因组座位的第一个编码外显子，构建成靶向载体，并将其电穿孔到胚胎干细胞。作为对照，将mCherry标记的人NMHC II-A-SV40 Polya导入同一基因座，以取代内源性NMHC II-A。在阳性(新霉素)和阴性(胸苷激酶)选择后，对候选胚胎干细胞克隆进行了分析。令人惊讶的是，在这两个案例中，24个胚胎干细胞克隆中有23个出现了同源重组。我们为每个构建选择了杂合克隆，并产生了嵌合小鼠，这表明了生殖系传播的证据。我们已经从杂合子小鼠的F1杂交中产生了少量的小鼠。到目前为止，还没有活的纯合子小鼠出生，NMHC II-B取代II-A。然而，与NMHC II-A切除的小鼠在胚胎6.5天(E)之前死亡不同，这些纯合子小鼠存活到E9.5，这表明表达非肌肉肌球蛋白II-B代替II-A可以挽救胚胎发育的早期阶段，如原肠形成。

Previous work from this laboratory showed that it is possible to replace one nonmuscle myosin heavy chain (NMHC) in mice with another using homologous recombination. For example, we replaced NMHC II-B with NMHC II-A resulting in expression of NMHC II-A under control of the endogenous II-B promoter. Presently, we are studying the effects of replacing murine NMHC II-A with GFP-tagged human NMHC II-B. The targeting construct was generated by introducing the GFP-NMHC II-B-SV40 polyA casette into the first coding exon of the NMHC II-A genomic locus and the construct was electroporated into embryonic stem cells. As a control, mCherry-tagged human NMHC II-A-SV40 polyA was introduced into the same locus to replace endogenous NMHC II-A. Following both positive (neomycin) and negative (thymidine kinase) selection, candidate embryonic stem cell clones were analyzed. Surprisingly, in both cases, 23 out of 24 embryonic stem cell clones showed homologous recombination. We selected heterozygous clones for each construct and generated chimeric mice which showed evidence of germline transmission. We have generated small numbers of mice from F1 crosses of heterozygous mice. To date, no live homozygous mice have been born with NMHC II-B replacing II-A. However, unlike the NMHC II-A ablated mice which die before embryonic day (E)6.5, these homozygous mice survive to E9.5, suggesting that expressing nonmuscle myosin II-B in place of II-A can rescue early embryonic stages in development, such as gastrulation.