Study Of The Transgenic Animal Model Using Sperm Vector

It has been studied in many ways in creating transgenic animals.However,choosing the most suitable gene transfering methods was the key to create.transgenic animal .The experiments studied on Japanese Big ear rabbit as the laboratory animal by using sperm vector method and analyzed, Its advantaye disadvantage,and the rate of success.Recombinant plasmid PLNCXHLFwas used in this study. The main component include promoter sequence (5' flank control rigon of goat P - lacto globulin gene of goat),foreign gene (cDNA of human lactoferrin gene),Marker gene (ampicillic gene) and genome of defective retrovirus. The following three methods were used to study transgenic animals. 1) Testis injection (transfecting the immature sperm of genital stem cell), 2) Deferent duct injection (transfecting the mature sperm),3) Implanted embryo after the sperm and foreign DNA were co-cultivated and fertilized in vitro. The results were1. After being by liposome, the recombinant plasmid were injected into testicular tissue of rabbit to infect genitals stem cell.The transgenic rabbits were hrough natural fertilization, altogether transgenic rabbits were generated in A,B,C,D,E and control group. DNA of tail tip was extracted 72 hours after the larva were delivered. The transgenic positive rate was found 35% and 28% when PCR detection were made with different primers which were designed according to foreign gene and selective gene respectively. Positive rate was relatively high, and the foreign gene could also maintain in testicular tissue for a long time, it could still be detected in the tissue in 7 months after being injected. However. Deferent results could be gained when the detection were made with different primers on different sites of the recombinant plasmid. This could be explained by transgene lost when the foreign gene recombinated and integrated in heterogenetic cell, or transgene degradated by cell nucleases.2. Matured sperm were infected by recombinant plasmid wrapped by liposome. These sperm were used in external fertilization, and four transgenic rabbits were obtainted by this way. Using different primers, PCR detection revealed the same result - high positive rate.3. The fertilization in vitro was carried out in this study. Immature ova from ovarian were cultured in vitro until they were in their maturation phrase. Mature sperm were extracted out from epididimises, and then transfected, capacitated in vitro. After external fertilization and being cultured in vitro, xygote had developed.