The Study Of CD59 And MCP Gene Transgenic Mouse And Swine By Sperm-Mediated Gene Transfer

On the base of the construction of recombinant human CD59 protein expression plasmids pEGFPN1-CD59 and human membrane cofactor protein (MCP) expression plasmids pEGFPN1-MCP, the transgenic mice were produced by sperm-mediated gene transfer. And the offsprings were detected by PCR, the perfusion into heart and RT-PCR. The transgenic pigs were produced by this method. Results were as follows:1. The plasmid pcDNA3-CD59,pcDNA3- MCP were digested by EcoRâ… and Kpnâ… . The fragments were connected with pEGFPN1. The recombinant human CD59 protein expression plasmids pEGFPN1-CD59 and human membrane cofactor protein (MCP) expression plasmids pEGFPN1-MCP wre constructed.2. The CD59 gene and MCP gene were mixed with liposome, then used to produce transgenic mice by 3 methods. Firstly, the gene/ liposome complex was directly injected into 5 male mice's testis. The 5 transfected males copulated with 25 females each 7 days later. And mices'embryos of various ages were gotten and 81 of 793 embryos had fluorescence. Secondly, we directly injected the gene/ liposome complex into 5 male mice's spermaduct and 75 of 810 embryos had fluorescence.Thirdly, we incubated the gene/ liposome complex with sperm, then artificially inseminated, and 53 of 357 embryos had fluorescence. The embryos which had fluorescence were detccted by PCR, and the PCR positive was 100%, this also means foreign gene can express at the early stage of embryo development.3. The transgenic mice lines were established by mating transgenic mice with none transgenic mice. The offsprings were identified by PCR, the perfusion of human blood serum and RT-PCR. In the offsprings, the CD59 positive rate was 41.2%, the MCP positive rate was 35.3%, the di-gene positive rate was 23.5%. The heartbeat time of F1 mice are obvious longer than these non-transgenic mice (P<0.05). After the RT-PCR detection of the total RNA from heart tissue, we found that the CD59 gene and MCP gene were expressed.4. The CD59 gene and MCP gene were mixed with liposome, then used to produce transgenic swine by 3 methods. Firstly, the gene/ liposome complex was directly injected into male swine's testis. The transfected males copulated with 8 females each 44 days later. And seven were pregnant (the pregnancy rate was 87.5%), and we got seventy-three piglets, three...