EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CE

为了研究非肌肉肌球蛋白II-A的功能，我们通过在基因外显子3插入新霉素抗性盒，建立了一只非肌肉肌球蛋白重链II-A(NMHC II-A)缺失的小鼠。此前，我们已经证明，消融非肌肉肌球蛋白重链II-B会导致小鼠心脏和大脑发育异常，死亡发生在胎龄12岁到出生当天。B-/-小鼠其他脏器正常，提示NMHC II-A可替代II-B。与NMHC II-B基因敲除相反，去除NMHC II-A导致了e7.5或更早的死亡，这表明NMHC II-A在早期胚胎发育中发挥了作用。NMHC II-A缺陷型胚胎比正常产仔小，组织严重紊乱。较小的体积似乎并不是由于细胞凋亡率的增加，这增加了较小的体积可能是由于细胞增殖减少。E7.5的野生型小鼠免疫细胞化学显示NMHC II-A存在于所有细胞层。然而，在这个阶段，内脏内胚层中没有NMHC II-B。NMHC II-A缺失小鼠的内脏内胚层严重紊乱，这表明NMHC II的两种亚型的缺失是导致缺陷的原因之一。胚胎干细胞同时表达NMHC II-A和II-B。NMHC II-A-/-ES细胞通过重新电穿孔NMHC II-A+/-细胞，然后在增加G418浓度的情况下进行筛选而产生。模拟早期发育阶段的类胚体已经被制造出来，并被检测是否存在胚胎细胞层的标记。虽然在NMHC II-A缺失型、杂合型和野生型拟胚体中存在一些内脏内胚层规范的标记，但在NMHC II-A缺失型、杂合型和野生型拟胚体中，一些内脏内胚层和中胚层发育的标记缺失或缺失。

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 early embryogenesis. NMHC II-A null embryos are smaller than normal littermates and are severely disorganized. The smaller size does not appear to be due to increased apoptosis, raising the possibility that the small size may be due to decreased cell proliferation. 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. NMHC II-A null mice have a severely disorganized 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. Embryoid bodies, which mimic the early stages of development, have been made and assayed for the presence of markers of embryonic cell layers. Although some markers of visceral endoderm specification are present in the NMHC II-A null, heterozygous and wild type embryoid bodies, a number of the markers of visceral endoderm and mesoderm development are missing or diminished in the null embryoid bodies.