【作者】 张庆；

【导师】 陈祥贵；

【作者基本信息】 西华大学 ， 食品科学， 2006， 硕士

【摘要】 Ercc61(excision repair cross-complementing rodent repair deficiency,complementation group 6-like)基因是在寻找胚胎发育相关基因的研究中,采用EST介导的基因克隆策略,从小鼠胚胎克隆的新基因。该基因长4002bp,定位于X染色体,最大开放阅读框(ORF)编码一1240个氨基酸组成的假定蛋白,属于SNF2家族ERCC6亚家族。表达谱分析提示该基因的功能可能与胚胎发育和肿瘤发生有关。初步研究结果提示Ercc61是小鼠胚胎发育相关基因,酒精致畸作用的分子靶点。本文主要在细胞学水平研究了基因Ercc61与P19胚胎癌细胞的关系。 研究目的:探讨基因Ercc61在P19胚胎癌细胞及P19分化细胞中的表达情况,并构建Ercc61的正、反义真核表达载体,在细胞学水平对基因功能进行初步研究。 研究方法:用一定浓度的二甲亚砜(DMSO)和维甲酸(RA)分别诱导处理P19胚胎癌细胞,使其分别向心肌细胞方向和神经细胞方向分化,提取P19细胞总RNA,RT-PCR检测P19细胞诱导分化前后Ercc61 mRNA的表达变化。用限制性内切酶Xho Ⅰ和Kpn Ⅰ双酶切含目的基因的pGEM-T-Ercc61载体,获得目的基因Ercc61序列,将其插入到真核表达载体pcDNA3.1(+)和pcDNA3.1(-)Myc His6多克隆位点的Xho Ⅰ和Kpn Ⅰ的酶切位点,相连构成基因表达质粒。将重组后的质粒进行细菌转化,先菌落PCR鉴定,然后质粒扩增、纯化后,再限制性内切酶Xho Ⅰ和Kpn Ⅰ酶切鉴定。以脂质体介导的方法体外瞬时转染正义载体至P19细胞,RT-PCR检测基因在细胞内的表达活性情况,并用MTT更多还原

【Abstract】 Ercc61 is a novel mouse development related gene which has been cloned using expressed sequence tags (ESTs) assembly. The cDNA of Ercc61 is 4002bp, encoding 1240 amino acids, mapped to mouse chromosome X. The novel gene belongs to ERCC6 subfamily of SNF2 family. Expression pattern analysis suggests that Ercc61 might implicate in embryo development and tumor genesis. The elementary study indicates that Ercc61 is a novel development related gene of mouse, as well as one of the molecular targets of alcohol teratogenic action. This paper mainly discuss the relationship between the novel gene Ercc61 and mouse embryonal carcinoma cells.Objective: The expression levels of Ercc61 in P19 embryonal carcinoma cells and differentiation cells of P19 are studied. Sense and antisense eukaryotic expression vectors of novel gene Ercc61 are constructed and its’ functions are studied in cell level.Methods: Mouse embryonal carcinoma cells were induced with DMSO and RA separately. Retinoic acid induced embryonal carcinoma cells to differentiate into neurons cells. In the presence of dimethylsulfoxide (DMSO), however, the cells differentiated to form cardiac elements. RT-PCR was performed to detect the expression changes of Ercc61 mRNA in the differentiation cells compared with in P19 cell. The ORF of Ercc61 was obtained by digestion with Xho Ⅰ and Kpn Ⅰ restriction endonuclease in the plasmid of pGEM-T-Ercc61. Recombinantvectors were constructed by inserting the target code gene into the MCS of plasmid pcDNA3.1(+) and pcDNA3.1(-) Myc His6 between Xho I and Kpn I sites. Then the recombinant plasmids were identified by colony PCR and restriction enzyme analysis. So we could choose appropriate recombinant plasmid which had been identified by restriction enzyme analysis to transfect P19 cells using lipofectin mediation. RT-PCR was performed to identify the expression levels of Ercc61 mRNA after transfection. Meanwhile, MTT assay was used to detect the proliferation of PI 9 cells which had been transfected. Mouse embryonal carcinoma cells were treated with certain concentration of cisplatin. Then RT-PCR was performed to identify the expression levels of Ercc61 mRNA in the cells which were treated with cisplatin. Proliferation of the mouse embryonal carcinoma cells was detected by using MTT assay.Results: Compared with control group, the expression activity of Ercc61 was not detected in mouse embryonal carcinoma cells. However the expression activity was significantly upregulated in the differentiation cells which induced by DMSO and RA separately. Cisplatin treatment presented no effect to expression of Ercc61. As screened by colony PCR and identified by restriction enzyme digestion, the sense and antisense expressin vectors were successfully constructed by subcloning the ORF of Ercc61 to pcDNA3.1.Conclusion: We infered that the novel gene Ercc61 might participate in the mouse embryonal carcinoma cell’s differentiation pathway. Cisplatin treatment presented no effect to expression of Ercc61. The sense and antisense eukaryotic expression vectors of novel gene Ercc61 were successfully constructed.更多还原