title: Inhibition of  vascular endothelial growth factor A expression in mouse granulosa cells by  lentivector-mediated RNAi.
abstract: Vascular  endothelial growth factor (VEGF) has been found responsible for the induction  of proliferation and differentiation in granulosa cells. We constructed four  short hairpin RNA (shRNA) expression plasmids targeting the mouse VEGFA gene,  and examined their effect on VEGF expression in mouse granulosa cells (MGC) in  vitro. Four different shRNA oligonucleotides targeting the coding sequence  of mouse VEGFA mRNA and one negative control (shNC) were designed and cloned  into a pGPU6/GFP/Neo siRNA expression vector, and transiently transfected into  MGC. At 48 h post-transfection, total RNA was extracted from the cells and  subjected to qRT-PCR analysis. The most effective interference vector, shVEGF1487  was chosen for lentiviral construction. The recombinant plasmid was then  transfected into 293FT cells via Lipofectamine(TM) 2000-mediated  gene transfer, for the production of lentivirus, and then concentrated via  ultracentrifugation. This lentiviral vector was then used for the transduction  of MGC. VEGFA gene expression, apoptosis genes and VEGFA receptor genes were  detected by qRT-PCR, the VEGFA protein level in culture media by ELISA assay  and protein levels in MGC by Western blot analysis. The four VEGFA expression  plasmids were successfully constructed and the most effective interference  vector, shVEGF1487, was chosen for lentiviral production and MGC transduction.  There was significant knockdown of the VEGFA gene, receptor genes and apoptosis  genes for all the shVEGF constructs, compared with the shNC and Mock controls.  The lentiviral vector also gave significant knockdown of the VEGFA gene.  Protein levels were lower for most of the shVEGFs based on Western blot  analysis with exception of VEGF1359; in this case, it was higher than shNC but  lower than for the Mock group. Lentivector-transduced MGC also gave lower  levels of protein. We conclude that shVEGF expression plasmids and lentivector  carrying RNAi are promising tools for the inhibition of VEGF, the corresponding  receptor genes, and apoptosis gene expression in MGC.
authors.affiliation: Department of Animal Breeding and Genetics, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China.
authors.forename: N
authors.initials: N
authors.lastname: Ally
authors.forename: X L
authors.initials: XL
authors.lastname: Zou
authors.forename: B C
authors.initials: BC
authors.lastname: Jiang
authors.forename: L
authors.initials: L
authors.lastname: Qin
authors.forename: L
authors.initials: L
authors.lastname: Zhai
authors.forename: P
authors.initials: P
authors.lastname: Xiao
authors.forename: H L
authors.initials: HL
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D005260:Female; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006107:Granulosa Cells / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A