Establishment of Haploid Cell Lines in Mammals and its Applications.

为建立哺乳动物单倍体细胞系，开展了以下实验。单倍体畸胎癌细胞的产生：小鼠未受精卵经孤雌激活后进行体外培养。将发育正常的胚泡注射到雄性小鼠的睾丸内。观察畸胎瘤和畸胎癌的产生。虽然注射正常受精卵可以获得很高的受精率(70%-80%)，但在单倍体胚胎中没有出现，可能是因为发育能力有限。单倍体胚胎干细胞的分离：将单倍体孤雌胚胎培养在含胎牛血清的饲养层细胞上。ES细胞的扩增非常少见，有时会出现分化细胞。它们虽然来源于单倍体胚胎，但仍是二倍体细胞。单倍体孤雌胚胎与正常受精胚胎的嵌合体分析：两只嵌合体小鼠进行…分析在大脑、眼睛和毛囊中有10-30%的孤雌生殖细胞，实际上对其他组织没有贡献。然而，几乎所有来自单倍体胚胎的细胞都是二倍体。在分析8-10天的嵌合胚胎时，孤雌生殖细胞的贡献率很高，在某些情况下，胚胎只由孤雌生殖细胞构成。说明孤雌生殖细胞在胚胎早期可以分化为各种类型的细胞。我们还发现，二倍体发生在着床早期。单倍体孤雌胚胎中癌基因的显微注射：通过显微注射将c-myc、Large T和E1a等癌基因导入单倍体孤雌生殖卵子。然后将卵子进行体外培养，并检测ICM的扩张情况。实际上，癌基因本身并没有观察到生长的改善。然而，在含有癌基因与增强子-启动子区域相连的DNA构建体的情况下，单倍体细胞的生长得到了改善。现在，我们认为通过显微注射癌基因来建立单倍体细胞系是可能的，这些癌基因被构建为在早期胚胎阶段发挥作用。较少

To establish a haploid cell line in mammal, the following experiments were carried out.1. Production of haploid teratocarcinoma cells : Mouse unfertilized eggs were parthenogenetically activated and cultured for in vitro. Normally developed blastcysts were injected into testes of male mice. Production of teratomas and teratocarcinomas was observed. Although high frequency (70-80%) of production was seen by injection of normal fertilized eggs, none were appeared in the case of haploid embryos, probably because of limited developmental ability.2. Isolation of haploid Embryonic Stem (ES) cells : Haploid parthenogenetic embryos were cultured on feeder cells in the medium containing fetal calf serum. Expansion of ES cells were very rare and differentiated cells were sometimes appeared. They were diploid cells in spite of their being derived from haploid embryos.3. Analysis of chimeric mice between parthenogenetic haploid embryos and normal fertilized embryos : Two chimeric mice were analyze … More d to have parthenogenetic cells in brain, eyes and hair follicles at 10-30% and actually no contribution to other tissues. However almost all cells derived from the haploidembryos were diploid. When analyzing 8-10 day chimeric embryos, contribution of the parthenogeneic cells were high and in some cases, embryos were constituted only by parthenogenetic cells. It demonstrated that the parthenogenetic cells can be differentiated to all cell types in early stage of embryos. We also found that diploidization was occurred at early implantation stage.4. Microinjection of oncogenes into haploid parthenogenetic embryos : Some oncogenes, such as c-myc, large T and E1A, were introduced into parthenogenetic haploid eggs by microinjection. The eggs were then cultured in vitro and expansion of ICM were checked. Actually no improvement on growth were observed by oncogenes themselves. However, in the case of DNA constructs that have oncogenes connected with enhancer-promoter region which functioned at pre- and postimplantation stage, improvement on haploid cell growth was observed. Now, we think it possible to establish the haploid cell lines by microinjection of the oncogenes which are constructed to function at early embryonic stages. Less