摘要
Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide frag-ments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2-CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results: DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion: The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.
Objective： To construct the small interfering RNA （siRNA） expression vector of carcino-embryonic antigen （CEA） and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods： Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide fragments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2- CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results： DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT- U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion： The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.
基金
the Main Project of Ministry of Education of China (No. 207150)
关键词
SIRNA
EC9706
胚胎抗体
抑制作用
EC9706
small interfering RNA （siRNA）
carcino-embryonic antigen （CEA）
