BPOZ基因纯合缺失ES细胞系的建立及其生长和分化研究

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.

Generation of ES Cell Clones Homozygous for BPOZ Gene Mutant and Analysis of Their Effect on Cell Growth and Differentiation

To investigate the effect of BPOZ gene mutant on cell growth or differentiation, ES cell clones which derived from BPOZ heterozygous mutant ES cells were cultured and selected in high concentration of G418,the genotype of the survival clones were identified by PCR. The expression of BPOZ gene in three type ES cells with different genotype were analyzed by RT-PCR, the expression of Oct3/4 gene was examined for identifying the differentiation state of the ES clones. The cell growth curve and 3H thymidine incorporation were performed to compare the growth rate and proliferation capability of the three type ES cells . Homozygous and wild-type ES cells were used to form teratoma in nude mice and embryoid bodies(EBs) in vitro for comparing their differentiation capability. The results showed that two undifferentiated ES cell clones homozygous for BPOZ gene mutant were obtained,in which the expression of BPOZ gene could not be detected. BPOZ gene mutant leads to ES cell growth suppression. ES cells homozygous for BPOZ gene mutant could differentiate to embryoid bodies in vitro and teratoma with cells or tissues from endoblast,mesoblast and ectoderm in nude mice. BPOZ gene knockout leads to ES cell growth suppression and has no significant influence on ES cell differentiation.