Research On EGFP Transgenic Chicken Mediated By Lentivirus

Lentivirus, as a member of the retrovirus family, have the ability to infect the dividingand non-dividing cells, and have been widely used to generate transgenic animals in recentyears. The domestic chicken has become an ideal model because of its oviparity, shortbreeding time, high efficiency of reproduction, and embryo obtained easily; and transgenesishas become an essential tool to sPFUdy avian developmental biology, behavioral biology andendocrinology. Therefore, developing a stable and efficient method to generate transgenicchicken would accelerate this process undoubtedly. This sPFUdy aimed at attempting to injectthe lentivirus into chicken testis to generate transgenic chicken. First, the lentiviruses werepackaged with4plasmids system and the packaging system was optimized. Then, thelentiviruses were injected into the new fresh embryo to generate transgenic chicken to test theactivity of the lentivirus. Next, the testicular cells were separated from the8-day-old chickenand infected with lentivirus after culPFUred for several time. Finally, the lentivirus wereinjected into the testis of20-day-old chicken, extracting the semen genome DNA for PCRanalysis after collecting semen when they grew to sexual maPFUrity. Chicken verifiedtransgenic sperm carrying were mated with normal female chicken to generate transgenicchicken. The results were as follows:1) the packaging efficiency was highest (titer was7.5×107PFU/ml) when the totalquantity of the four plasmids was50μg for90mm petri dish (the quantity of the four plasmidspMDL, pRSV-Rev, pMDG and pCD513B-1was10μg,10μg,10μg,20μg respectively) andthe pH of the HN Buffer was7.05in calcium phosphate precipitation method.2) The lentiviruses aforesaid were injected into the subgerminal cavity of170new freshfertilized eggs, and there were21chicks (21/170,12.4%) after hatching.6(6/21,28.8%) ofthem were verified to be positive after extracting the tissues genome DNA for PCR analysis,and3(3/21,14.4%) of them were detected the GFP after extracting the related tissues totalprotein for Western Blot analysis.3) Testicular cells were separated from8-day-old chicken with the two-step enzymedigestion method, and were purified preliminarily with multiple differential attachmentmethod. Infecting the testicular cells with lentivirus aforesaid when the cell density was70% above, and the final infecting efficiency was about5%.4) Lentiviruses aforesaid were injected into the testis of1520-day-old chicks, extractingthe semen genome DNA for PCR analysis after collecting semen when they grew to sexualmaPFUrity,2(2/15,13.3%) of them were detected to be positive. Then, mating the two semenpositive chicken with normal female chicken to generate their offspring, during the38offspring of one chicken, there was only1chick (1/38,2.63%) that verified the target gene,but no GFP expression was detected.This sPFUdy has optimized the packaging system, and demonstrated again that lentivirusembryo injection approach was an efficient way to generate transgenic chicken. Althoughtransgenic chicken was not obtained through lentivirus testis injection approach, this sPFUdyhas proved preliminarily that foreign gene could ben integrated into spermatogonial stem cells(SSCs) via lentivirus vector, and passing to offspring by sperm. This shed some light togeneration of other transgenic animals.