Construction of porcine CCK pDNA and its expression in COS-7 cells.
CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamin 2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.
Bai
Jigang
J
Department of General Surgery, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Lü
Yi
Y
Bai
Qiaoling
Q
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002766:Cholecystokinin / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002766:Cholecystokinin