EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

为了研究非肌肉肌球蛋白II-A的功能，我们通过将新霉素抗性盒插入到外显子3的基因中来创建非肌肉肌球蛋白重链II-A（NMHC II-A）敲除小鼠。以前，我们已经表明，消融小鼠的非肌肉肌球蛋白重链II-B导致心脏和大脑发育异常，致死性发生在E12和出生之日之间。 B-/-小鼠的其他器官表现正常，表明NMHC II-A可以替代这些器官中的II-B。 与NMHC II-B敲除相反，NMHC II-A的消融在E7.5或更早时导致死亡，表明NMHC II-A在原肠胚形成期间的作用。在E7.5野生型小鼠的免疫细胞化学显示在所有细胞层中存在NMHC II-A。 然而，在这个阶段，NMHC II-B是缺席的内脏内胚层。 NMHCII-A基因敲除小鼠缺乏或具有严重发育不全的内脏内胚层，这表明NMHC II的两种亚型的缺乏促成了缺陷。 NMHC II-A-/- ES细胞通过NMHC II-A+/-细胞的再电穿孔，随后在增加浓度的G418下选择而产生。 仅表达NMHC II-B的NMHC II-A-/- ES细胞似乎以比NMHC II-A+/-或野生型细胞更快的速率增殖，并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.