Construction Of Recombinant Lentiviral Vectors For Goat Chop Gene Overexpression And Its Interference RNA

C/EBP homologous protein （CHOP） was first identified to be a member of theCCAAT/enhancer binding proteins （C/EBPs） that serves as a dominant negative inhibitor ofC/EBPs.CHOP is also known as growth arrest-and DNA damage inducible gene153（GADD153）, DNA–damage-inducible transcript3（DDIT3） and C/EBPζ.Our previous studiesshowed that CHOP was specificly expressed in atresia ovaries compared to with normalovaries. Howerver, the role of CHOP in the development of reproduction have not beendocumented before. In this study, we cloned CDS of goat chop gene and constructed chopover-expression and shRNA recombinant lentiviral vectors and to have a better understandingof the mechanism of chop during follicular atresia. According to Ovis aries （AY943948）, Bostaurus （NM₀01078163.1）,the full-length CDS sequence of goat chop is cloned, chop CDSwas cloned into the shuttle vector pCD513B for the construction of pCD513B-chop. Threeinterfering sequence targeting chop and a negative sequence were designed, synthesized andinserted into plasmid pCD513B-U6lentiviral vector. The viral stock was prepared byco-transfection of shuttle plasmids and the packaging plasmid mix to HEK293T cells. HEK293T cells were then transduced with an appropriately diluted lentivirus supernatant for thetitration of virus titer. After infection of EEC cells with these lentiviruses, the expression ofchop was detected by Real-time PCR. The results are as follows:1.We designed a pair of primers and clone the CDS of goat chop through RT-PCR.The1.0%agarose gel electrophoresis results of PCR showed the669bp gene fragment wasobtained from goat ovary total RNA and amplification with a pair of special primers designedat first. The sequence of chop gene obtained was sequencing and reported toGenBank（KC188779.1）. The goat CHOP has an open reading frame of669bp coding sequences whichencoded168amino acids. From the deduced amino acid sequence, the molecular weight （Mw） and theoretical isoelectric point （pI） of the goat CHOP were53755.9and5.01, respectively. Goat CHOP hadhighest homology with that of ruminant Ovis aries （99%） and Bos Taurus （97%）.2.After being digested and identified by restrictive endonuclease, pCD513B waslinearized. Chop gene was connected into pCD513B vector and renamed the expressionvector pCD513B-chop. The results of1.0%agarose gel electrophoresis of recombinant pCD513B-chop digested by restrictive endonuclease showed special bands of8100bp and700bp. It confirmed that the pCD513B-chop was constructed successfully.3.Four Pairs of small hairpin oligonucleotide targeting ORF of goat chop gene weredesigned and synthesized. The oligonueleotides were annealed to produce complementarydouble-strand as shRNA gene and then cloned into the lentiviral-vectorpCD513B-U6-shRNA-chop. The2.0%agarose gel electrophoresis results of PCR showed the350bp gene fragmentwas obtained by amplifying with a pair of specialprimers designed. Itconfirmed that the pCD513B-U6-shRNA-chop were constructed successfully.4.The correct reeombinant lentiviral-vector plasmid and packing mixer wereco-transfeeted into HEK293T cells, then reeombinant lentiviruses were produced and the titerof virus was5-8×107TU/ml.EEC cells infected with lentiviral vector carrying full-lengthchop gene successfully expressed high-level chop. The mRNA of CHOP reduced to less than60%after delivery of lentiviral vector carrying shRNA sequence.The lentiviral vectors carrying chop gene and shRNA sequence targeting goat chop havebeen successfully constructed．It has laid an experimental basis on the follicular atresia and itsfunctional study．...