Construction of recombinant adenovirus Ad-rat PLCg2-shRNA and successful suppression of PLCg2 expression in BRL-3A cells.
Phospholipase Cg2 (PLCg2) induces apoptosis of immune and tumor cells; however, it remains unclear whether PLCg2 promotes hepatocyte apoptosis during liver regeneration (LR). Therefore, to establish a framework for further exploring the function of PLCg2, we generated recombinant adenoviruses carrying a template encoding short hairpin (sh)-RNA targeting PLCg2 (Ad-PLCg2-shRNA), which were used to silence the expression of PLCg2 in BRL-3A cells. First, three pairs of PLCg2-shRNAs were designed, synthesized, and cloned into a shuttle vector, pHBAd-U6-GFP, after annealing. The recombinant shuttle plasmids were co-transfected with the backbone vector pHBAd-BHG into HK293 cells to package the recombinant Ad-PLCg2-shRNAs used to infect BRL-3A cells. Infection efficiency was monitored by observing the number of GFP-positive cells under a fluorescent microscope. To determine the recombinant adenoviruses with the highest silencing efficiency, levels of PLCg2 mRNA were evaluated by qRT-PCR. DNA sequencing confirmed that the correct shRNA coding sequences were inserted into the shuttle vectors and adenoviral plasmids. The titers of three recombinant adenoviruses were at least 1 x 10(10) PFU/mL. The most effective adenoviral construct, with interference efficiency of 77%, was determined by qRT-PCR. These results show that a recombinant adenovirus, Ad-PLCg2-shRNA, was developed and was effective at silencing the rat PLCg2 gene. This construct may contribute to the study of PLCg2 in hepatocyte apoptosis during LR.
Animal Science and Technology School, Henan University of Science and Technology, Luoyang, Henan Province, China.
X G
XG
Chen
Animal Science and Technology School, Henan University of Science and Technology, Luoyang, Henan Province, China.
Q X
QX
Lv
Animal Science and Technology School, Henan University of Science and Technology, Luoyang, Henan Province, China.
X Q
XQ
Zhou
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D008115:Liver Regeneration / Q000235:genetics*; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051966:Phospholipase C gamma