Construction Of Recombinant Lentiviral Vectors For Mouse Zhangfei Gene Expression And Its Interference RNA

Zhangfei also known as CREBZF or SMILE, is a member of CREB/ATF protein family.Zhangfei has been considered mainly as a basic leucine zipper (bZIP) transcription factor, incoordination with other transcription factor, play a role in the latent infection of HerpesSimplex Virus-1(HSV-1), apoptosis and the mammalian endoplasmic reticulum stress (ERS)and unfolded protein response. Our previous studies showed that Zhangfei was specificlyexpressed in atresia ovaries compared to with normal ovaries. Howerver, the role of Zhangfeiin the development of reproduction have not been documented before. In this study, weconstructed zhanagfei shRNA expression and over-expression recombinant lentiviral vectorsand to have a better understanding of the mechanism of Zhangfei during follicular atresia.According to GenBank, the full-length cDNA sequence of mouse Zhangfei, Zhangfei cDNAwas cloned into the shuttle vector pCD513B for the construction of pCD513B-Zhangfei.Three interfering sequence targeting Zhangfei and a negative sequence were designed,synthesized and inserted into plasmid pCD513B-U6lentiviral vector. The viral stock wasprepared by co-transfection of shuttle plasmids and the packaging plasmid mix to HEK293cells. HEK293cells were then transduced with an appropriately diluted lentivirus supernatantfor the titration of virus titer. After infection of NIH3T3cells with these lentiviruses, theexpression of Zhangfei was detected by RT-PCR and Western Blotting. The results are asfollows:(1) The1.0%agarose gel electrophoresis results of PCR showed the1400bp genefragment was obtained from mouse ovary total RNA and amplification with a pair of specialprimers designed at first. The sequence of Zhangfei gene obtained was consistent with thereported, and had no mutation. The construction of lentivirus vector, When sequencing iscorrect. After being digested and identified by restrictive endonuclease, pCD513B waslinearized. Zhangfei gene was conected in to pCD513B vector and renamed the expressionvector pCD513B-Zhangfei. The results of1.0%agarose gel electrophoresis of recombinantpCD513B-Zhangfei digested by restrictive endonuclease showed special bands of8100bpand1400bp. It confirmed that the pCD513B-Zhangfei was constructed successfully.(2) Five Pairs of small hairpin oligonucleotide targeting ORF of Zhangfei gene weredesigned and synthesized. The oligonueleotides were annealed to produce complementary double-strand as siRNA gene and then cloned into the lentiviral-vectorpCD513B-U6-shRNA-Zhangfei. The2.0%agarose gel electrophoresis results of PCR showedthe350bp gene fragmentwas obtained by amplifying with a pair of specialprimers designed.It confirmed that the pCD513B-U6-shRNA-Zhangfei was constructed successfully.(3) The correct reeombinant lentiviral-vector plasmid and packing mixer wereco-transfeeted into HEK293cells, then reeombinant lentiviruses were produced and the titerof virus was5~10×107TU/ml.(4) NIH3T3cells infected with lentiviral vector carrying full-length Zhangfei genesuccessfully expressed high-level Zhangfei. The mRNA of Zhangfei reduced to less than70%after delivery of lentiviral vector carrying shRNA sequence.The lentiviral vectors carrying the full length Zhangfei gene and shRNA sequencetargeting Zhangfei have been successfully constructed．It has laid an experimental basis on thefollicular atresia and its functional study．...