Study Of Expression Of Novel Gene Ercc6l In P19 Embryonal Carainoma Cell

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 â…  and Kpn â…  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.