Antiapoptotic gene BAG-1 vector structure of RNA interference and endogenous targeted screening in colon cancer cell lines.
The purposes of the present work were to construct the shRNA plasmids for BAG-1 gene of human and test the expression of mRNA and protein of BAG-1. Recombinant plasmids containing green fluorescent protein reporter genes are constructed using gene cloning methods. The shRNA plasmids for the BAG-1 gene are constructed by RNA interference technology. We applied fluorescent plasmid-transfected target cells in the cell transfection experiments and monitored the transfection rate of plasmids by observing the fluorescence amount. We transfected three synthesized shRNA in target screening cell and adopted RT-PCR and Western test to identify the difference of target gene transfection and translation level in cells. The specific shRNA plasmid for the BAG-1 gene was successfully recombined, and stably transfected colon cancer Lo Vo cell lines were obtained. The results present that the constructed shRNA plasmids significantly inhibited the expression of mRNA and protein of Lo Vo cell BAG-1, and can maintain the effect for a long term. pGPH1/GFP/Neo-BAG-1-homo-825 was screened as the optimum sequence of interference so as to lay a solid foundation to explore into the research on the BAG-1 gene and the biological behavior of colon cancer cells. It showed the remarkable advantage of RNAi in the generation of posttranscriptional gene silencing.
Department of General Surgery, Qilu Hospital of Shandong University, Jinan, 250012, China.
Nian-feng
NF
Sun
Ai-ling
AL
Tian
Zhan-ao
ZA
Liu
San-yuan
SY
Hu
An-bin
AB
Hu
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D014162:Transfection
C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins