Expression Of MxA In SSCs Mediated By Lentiviral Vector And Its Transplantation In Vivo

Objective: MxA gene was first reconstructed into the lentiviral vector, packaged by the 293FT cells, and then infected into the spermatogonial stem cells (SSCs) both in vivo and in vitro. The SSCs which has been infected by the lentivirus was transplanted into the testis of the receptor chicken, and the distribution and expression of MxA gene was detected in testis. The DNA of the semen was collected to detect the expression level of MxA in sperm. The sperm which contained the target gene can be selected to perform the insemination in order to produce the transgenic chicken.Methods: TOPO cloning technique was selected to construct the lentivirus vector: pLenti6/V5-EGFP-MxA. PCR reaction testified the EGFP-MxA gene was located in the correct direction without any gene rearrangement. The eukaryotic cell 293FT, after transfected by the reconstructed lentivirus, can express the specific green fluorescence with granulose light distributing in the cytolymph. After cotransfection, we harvest the reconstructed lentivirus whose titer was determined before we use it to infect the target cells. SSCs was isolated from the testis of 25d cock and cultured until the cell density was proper for the infection. Meanwhile, the sexual maturity cock was injected with busulfan in order to degrade its endogenetic SSCs level. After the transplantation of SSCs into testis, the RT-PCR reaction were operated to detect the transcriptional level of target gene and frozen section of testis tissue was performed for the analysis of green fluorescence detection and immunohistochemistry of the distribution and expression of MxA gene. Finally, the semen was harvested to extract its DNA and detect the expression level of target gene in the sperm of the acceptor cock. This research also included the infection of SSCs in vivo, by which we directly injected the recombinant virus into the testis of sexual maturity cock, same methods were selected to detect expression of target gene.Results:1. Both the PCR reaction and restriction enzyme analysis demonstrated that the recombinant lentivirus vector was successfully constructed.2. Optimal parameter for the operation of virus packaging conditions were :a. Optimal quantity proportion of the vector, packaging mix and lipofectamine: vector plasmid : packaging mix : lipofectamine(wt/vol) = 1 : 3 : 4b. Optimal inoculum density of the packaging cells 293FT: 1×105 cells/mlc. Optimal culturing temperature for packaging cells: 37℃d. Optimal time for harvesting recombinant virus: 48h post infection.3. The titer of recombinant virus was concentrated by ultrafiltration to a level of 107 cfu/ml.4. The specific green fluorescence with granular light distributing in the cytoplasm could be detected by the analysis of SSCs after infection through fluorescence microscope observation. RT-PCR reaction and immunohistochemistry analysis verified the expression of fusion gene in SSCs at the level of transcription and translation. After transplantation into the testis of acceptor roosters, the tissue RT-PCR and frozen section immunohistochemistry also demonstrated the positive result of the target gene expression. While analyzing the sperm of acceptor roosters after transplantation, we successfully detected the MxA gene by PCR reaction.5. In the study of expression of MxA in SSCs in vivo, the MxA gene could only be detected by the analysis of RT-PCR reaction and immunohistochemistry. There showed no positive result when analyzing the DNA of sperm in infected sample.Conclusions: The method that choosing SSCs as gene delivery vector which mediate target gene expressing in the acceptor sample showed a higher efficiency compared with the infection in vivo in which the target gene was directly injected into the tissue of sample. This result will lay the foundation for the further understanding of producing anti-virus transgentic chicken by combinating the methods of transfecting the SSCs by anti-virus gene MxA and transplantation technique.