Detection Of BMP-8a And BMP-8b In Mouse Granulosa Cells, And Construction Of The RNA Interference Vector In Ovine BMPR-IB Gene Targeting

The objective of this study was to study the expressions of BMP-8a and BMP-8b in mousegranulose cells, for more in-depth understanding of follicular development process; and constructing theRNA interference vector in targeting ovine BMPR-IB, which will be used in research on how theexpression of BMPR-IB from granulosa cells in vivo influences the fecundity traits in sheep. This studyincluded two expriments:The first expriment of this study was to detect the expressions of BMP-8a and BMP-8b in mousegranulosa cells with RT-PCR and sequencing. Six21-23day-old female mice were hypodermicallyinjected with10IU of PMSG. After48hours, the bilateral ovaries were taken and the granulose cells wrecollected by piercing the ovarian follicles. The granulose cells were incubated in DMEM/F12supplemented with10%FBS,100IU/ml Penicillin and Streptomycin at37℃,5%CO2in a carbondioxide incubator. After5days, the cells were collected and total RNA was extracted with Trizol. Theexpressions were analysised by RT-PCR and sequencing.The result showed that mouse granulosa cellscan express BMP-8a transcritpt variant2and BMP-8b.The second expriment of this study was to construct the RNA interference vector in targetingBMPR-IB in sheep. Eight interference sequences were designed based on BMPR-IB mRNA sequence（accession: NM₀01009431） by using Dharmacon siDESIGN software. The siRNAs were designed forsense shRNA and antisense shRNA. Eight groups of sense shRNA and antisese shRNA were annealedand inserted into the PLVX-shRNA vector. The reconstructed vectors were then transformed to DH5αcompetent cells which were cultured in LB medium and positive clones were selected on the solidmedium. The inserts were validated by EcoRI and BamHI double enzymatic digestion, HindⅢ enzymedigestion and sequencing.