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研究BPOZ基因缺失对细胞生长和分化的影响.以高浓度的G418筛选BPOZ基因杂合缺失型ES细胞,PCR鉴定抗高浓度G418细胞克隆基因型;半定量RTPCR分析3种基因型ES细胞BPOZ基因的表达情况,分析3种基因型ES细胞Oct34基因的表达以明确ES细胞分化状态.利用3种基因型ES细胞进行细胞生长曲线和3H胸嘧啶核苷参入实验比较其生长速度和增殖能力.以裸鼠荷瘤实验和类胚体形成实验比较BPOZ基因纯合缺失型ES细胞与野生型ES细胞生长分化能力.结果表明,筛选获得两个BPOZ基因剔除的纯合ES细胞克隆;筛选得到的纯合ES细胞中BPOZ基因表达完全缺失,细胞处未分化状态.与野生型ES细胞相比,BPOZ基因纯合缺失型ES细胞生长受抑,增殖能力减弱.BPOZ基因纯合缺失型ES细胞可分化形成类胚体和具备来自3个不同胚层的细胞和组织的畸胎瘤.BPOZ基因剔除使ES细胞生长受抑,对ES细胞分化发育没有明显影响.
The effects of BPOZ gene deletion on cell growth and differentiation were studied. BPOZ gene-deficient ES cells were screened with high concentration of G418, and the cloned g418 genotypes were identified by PCR; semi-quantitative RT-PCR was used to analyze the BPOZ gene of three genotype ES cells. The expression status of the three genotype ES cells was analyzed to determine the differentiation state of ES cells. The cell growth curve and 3H thymidine incorporation experiment were compared using 3 genotype ES cells to compare their growth rate and proliferation ability. The ability of BPOZ gene homozygous deletion type ES cells and wild type ES cells to grow and differentiate was compared between tumor bearing experiments and embryoid body formation experiments in nude mice. The results showed that two homozygous ES cell clones with BPOZ knockout were obtained; The expression of BPOZ gene was completely deleted in ES cells, and the cells were in an undifferentiated state. Compared with wild-type ES cells, the growth of homozygously deleted ES cells of BPOZ gene was inhibited and the proliferation ability was weakened. The BPOZ gene homozygous deletion-type ES cells can be differentiated. Formation of embryoid bodies and teratomas with cells and tissues from three different germ layers. BPOZ knockout inhibited the growth of ES cells and had no significant effect on the differentiation and development of ES cells.