The Function of Nonmuscle Myosin Heavy Chains

该实验室以前的工作表明，有可能使用同源重组将小鼠中的一条非肌肉肌球蛋白重链（NMHC）替换为另一条。 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替代鼠NMHC II-A的效果。 通过将GFP-NMHC II-B-SV 40 polyA盒引入NMHC II-A基因组基因座的第一编码外显子中来产生靶向构建体，并将构建体电穿孔到胚胎干细胞中。 作为对照，将mCherry标记的人NMHC II-A-SV 40 polyA引入相同的基因座以替代内源性NMHC II-A。 在阳性（新霉素）和阴性（胸苷激酶）选择后，分析候选胚胎干细胞克隆。 令人惊讶的是，在这两种情况下，24个胚胎干细胞克隆中有23个表现出同源重组。 我们为每个构建体选择杂合克隆并产生嵌合小鼠，其显示生殖系传播的证据。 我们已经从杂合小鼠的F1杂交中产生了少量的小鼠。 到目前为止，没有活的纯合子小鼠出生时NMHC II-B取代II-A。 然而，与NMHC II-A消融小鼠在胚胎日（E）6.5前死亡不同，这些纯合子小鼠存活至E9.5，表明表达非肌肉肌球蛋白II-B代替II-A可以挽救发育中的早期胚胎阶段，如原肠胚形成。

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.