Construction of a mammalian cell expression vector pAcGFP-FasL and its expression in bovine follicular granulosa cells

摘要

Fas ligand (FasL) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the Fas. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis.?To reveal?the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary,?the Fas ligand gene was cloned using RT-PCR.?By deleting the stop codon, the amplified Fas ligand gene was directionally cloned in frame into the eukaryotic expression vector pAcGFP-N1. The pAcGFP-bFasL recombinant plasmid was then transfected into bovine follicular granulosa cells by using lipofectamine 2000. Expression of AcGFP was observed under fluorescent microscopy and the transcription and expression of?Fas ligandwas detected by RT-PCR and Western-blot. The results show that the pAcGFP-bFasL recombinant plasmid was successfully constructed. AcGFP expression was detected as early as 24 h after transfection and the percentage of AcGFP positive cells reached about 68%.?As expected, a 847 bp fragment was amplified by RT-PCR and a 59 kD target protein was detected by Western-blot from the transfected cells. This study will thus serve as a valuable tool in understanding the mechanism of regulation of?Fas ligand?on bovine oocyte formation and development.