Construction of recombinant adenovirus Ad-rat PLCg2-shRNA and successful suppression of PLCg2 expression in BRL-3A cells.
Construction of recombinant adenovirus Ad-rat PLCg2-shRNA and successful suppression of PLCg2 expression in BRL-3A cells.
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DOI：
10.4238/gmr.15028029
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发表时间：
2016-05
期刊：
影响因子：
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通讯作者：
X. Chen;Q. Lv;X. Q. Zhou
中科院分区：
文献类型：
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作者：
X. Chen;Q. Lv;X. Q. Zhou
磷脂酶CG2(PLCG2)可诱导免疫细胞和肿瘤细胞的凋亡，但目前尚不清楚PLCG2是否促进肝再生过程中肝细胞的凋亡。因此，为了建立进一步研究PLCG2功能的框架，我们构建了携带针对PLCG2的短发夹(Sh)-RNA的重组腺病毒(Ad-PLCG2-shRNA)，用于沉默PLCG2在BRL-3A细胞中的表达。首先，设计、合成了三对PLCG2-shRNAs，并将其克隆到穿梭载体pHBAd-U6-GFP中，经处理后构建成穿梭载体pHBAd-U6-GFP。将重组穿梭载体与骨架载体pHBAd-BHG共转染HK293细胞，包装用于感染BRL-3A细胞的重组Ad-PLCG2-shRNAs。通过在荧光显微镜下观察GFP阳性细胞数来监测感染效率。为了确定沉默效率最高的重组腺病毒，采用qRT-PCR方法检测PLCG2mRNA的表达水平。DNA测序证实正确的shRNA编码序列已插入穿梭载体和腺病毒载体。3株重组腺病毒的滴度均在1×10(10)pfu/mL以上。通过qRT-PCR确定了最有效的腺病毒载体，干扰效率为77%。这些结果表明，重组腺病毒Ad-PLCG2-shRNA成功地沉默了大鼠PLCG2基因。为进一步研究PLCG2在肝细胞凋亡中的作用奠定了基础。
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.