Lentivirus-Mediated Expression of MxA in Chicken Spermatogonial Stem Cells

Abstract

The aim of this study was to construct a lentivirus vector that carries the human myxovirus-resistant A (MxA) anti-virus gene for efficiently infecting rooster spermatogonial stem cells (SSCs). A lentiviral vector carrying the enhanced green fluorescent protein (EGFP) and MxA fusion gene (EGFP-MxA) was constructed using TOPO technology in this study. Enhanced green fluorescent protein-MxA was inserted in the right orientation as determined by restriction enzyme digest. No gene recombination in the vector occurred. After infecting 293FT cells, EGFP-MxA fusion protein was expressed as granular green fluorescence characteristic of EGFP-MxA expression, suggesting that the TOPO expression vector was properly constructed and the fusion protein expressed correctly. The EGFP-MxA recombinant lentivirus was packaged by cotransfecting 293FT cells with EGFP-MxA and the packaging plasmids. We also purified SSCs from testicle tissues from 25-day-old roosters for infection with the EGFP-MxA recombinant virus. After infecting rooster SSCs with the recombinant virus for 72 h, EGFP-MxA expression was detected by EGFP expression. Enhanced green fluorescent protein-MxA expression in SSCs was further confirmed at the transcription level by RT-PCR and at the protein level by immunocytochemistry. Staining with Hoechst 33342 showed that infected SSCs differed from the sertoli cells. Similar to stem cells, SSCs were positive for alkaline phosphoric acid enzyme and for stage-specific embryonic antigen-1 stem cell factor. The results demonstrated that the recombinant virus made in our study can infect rooster SSCs efficiently to express the anti-virus protein MxA, establishing the basis of transferring MxA into SSCs to obtain virus-resistant, genetically-modified roosters.