EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

为了研究非肌球蛋白II-A的功能，我们通过在基因外显子3处插入新霉素耐药盒，建立了非肌球蛋白重链II-A （NMHC II-A）零小鼠。先前，我们已经表明，小鼠非肌球蛋白重链II-B的消融导致心脏和大脑发育异常，并在12岁至出生当天发生致命性。B-/-小鼠的其他器官显示正常，提示NMHC II-A可以在这些器官中替代II-B。与NMHC II-B敲除相比，NMHC II-A的消融导致E7.5或更早的死亡，这表明NMHC II-A在原肠形成过程中起作用。E7.5时野生型小鼠免疫细胞化学显示NMHC II-A在各细胞层均存在。然而，在这一阶段，内脏内胚层中没有NMHC II-B。NMHCII-A缺失的小鼠缺乏或具有严重不发达的内脏内胚层，这表明NMHCII的两种亚型的缺失导致了这种缺陷。胚胎干细胞同时表达NMHC II-A和II-B。通过重新电穿孔NMHC II-A+/-细胞，然后在增加浓度的G418下选择，生成NMHC II-A-/- ES细胞。仅表达NMHC II-A-/- ES的细胞比NMHC II-A+/-或野生型细胞增殖速度更快，且不形成胚胎干细胞集落。

In order to investigate the function of nonmuscle myosin II-A, we created a nonmuscle myosin heavy chain II-A (NMHC II-A) null mouse by inserting the neomycin resistance cassette into the gene at exon 3. Previously, we have shown that ablation of nonmuscle myosin heavy chain II-B in mice results in abnormalities in the development of the heart and brain with the lethality occurring between E12 and the day of birth. The other organs of the B-/- mice appear normal, suggesting that NMHC II-A could substitute for II-B in these organs. In contrast to the NMHC II-B knockout, ablation of the NMHC II-A resulted in lethality at E7.5 or earlier, suggesting a role for NMHC II-A during gastrulation. Immunocytochemistry of wild type mice at E7.5 showed the presence of NMHC II-A in all cell layers. At this stage, however, NMHC II-B was absent from the visceral endoderm. NMHCII-A null mice are lacking or have a severely underdeveloped visceral endoderm suggesting that the absence of both isoforms of NMHC II contributed to the defect.Embryonic stem (ES) cells express both NMHC II-A and II-B. NMHC II-A-/- ES cells were generated by re-electroporation of NMHC II-A+/- cells followed by selection at increased concentrations of G418. NMHC II-A-/- ES cells which only express NMHC II-B appear to proliferate at a faster rate than NMHC II-A+/- or wild type cells and do not form embryonic stem cell colonies.