Production Technique Of Efficient Transgenic Chickens Delivered By Lentiviral Vectors

Applications of transgenic chicken model system may include expression of functional proteins in eggs as bio-reaction, poultry breeding and basic research in molecular and developmental biology. In this present study, we atemped to construct the system of production technique of transgenic chickens delivered by leniviral vectors, primary product the transgenic chickens which expressing the pharmic protein of tissue plasminogen activateor (tPA) and set up the system to select appropriate oviduct special expressing promoter, to prepare to product the oviduct bio-reaction and transgenic breeding.Cells derived from different species with dissimilar differentiational states including Hela, 293FT, C127, chicken embryo fibroblasts(CEF) and chicken primary germ cells(cPGC) were infected in vitro with recombinant lentiviral vectors based on HIV-1 which were marked with enhance green fluorescence proteins (EGFP) gene. Data showed that lentivial vectors could infect all types cells including normal or cancer cells and primary culture cells or infinite cell lines with higher level of expression. The efficiency of transient expression in Hela and 293FT is about 10⁸TU/ml, 5×10⁷TU/ml in C127, 10⁶TU/ml in CEF and 10²TU/ml in cPGC. Transfaction of lentiviral may induce adverse morphological transform of cells in vitro. Using this lentiviral vectors to inject fresh lay eggs of hens, we have got EGFP transgenic chickens with the level that the signal of green fluorescence can be direct found. A total of 204 eggs were injected with lentivirus in our two experiments from which 30 (15%) chicken hatched. 16 out of 30 (53%) chickens were determined to contained vector sequences by DNA dot blotting analysis and PCR using EGFP specific primers. After chicks raised to sexual maturity, 10 offspring samples of 3 Go cockerels mated with wild-type hens were analyzed by DNA dot blotting analysis. Two G₁ offspring were transgenic suggested exogenous gene were allowed to transmit through the germ line. 4 of 16 can be found to emit green fluorescence signal from appearance of live chicken with remarkable phenomenon of mottled expression in their various parts such as toe, beak and naked skin in face, claw and ear. In the GFP-positive birds, tissues were direct observed intense EGFP expression in small intestine, rectum and liver. However the level of GFP expression was not homogeneous between different chicken even in the same organs. Expression analysis of GFP in sections of tissues from transgenic chickens detected expression in the skin, liver, pancreas, testis, intestine, blood vessel. More important in testis sections the transgene was expressed at levels detectable by direct fluorescence focusing on seminiferous tubules using this lentiviral vector. We also examined the feasibility via LM-PCR to analysis exogenous genes integrating sites. These results indicate that HIV-based lentiviral vectors can be easy to use to efficiently generate transgenic birds with with high efficiency, low-cost manipulation and simple apparatus.Based on above researches, we constructed the lentivial vextors expressing human tissue plasminogen activateor, in which the tPA and the GFP proteins were fused in Asp-Pro formic acid sensitivity site. A total of 34 chicken embryos were injected with this lentivirus, from which 7 (20.6%) chicken hatched. 2 out of 7 chickens were found integration of exogenous genes by DNA dot blotting analysis and PCR using EGFP specific primers. The two transgenic chickens are all cockerels. Although no green fluorescence sigle was detected in these two transgenic chickens, the target protein was also be found the weak expression byimmunofluorescence analysis in pancreas. The tPA activity was found in pancreas and liver by fibrin plate analysis.The production of pharmic protein as bio-reaction of Poultry is regarded due to the character that special/efficient expressing in eggs. To fulfil this aim, it is important to select appropriate promoter, so we cloned the whole sequences of SV40 large antigen (TAG) from 293FT cells, and constructed the lentiviral vectors expressing TAG with neo fastness. After producting virus, the virus was used to infect the oviduct epithelia of newly laying hen to construct immortalized oviduct epithelia. The immortalized cells have a good proliferation and can be used to estimate the transcription efficience of different promoters. Two lentiviral vectors with blasticidin fastness were constructed which have different ovalbumin (OV) promoters, OV1345 and OV2964. OV2964 have the first intron of OV gene, but not in OV1345. After the immortalized oviduct epithelia was infected with the difference virus and selected by antibiotic, the DNA and mmRNA were extracted and were analysis by relative quantitative FCM-PCR. The data showed that integration efficient of the virus with OV1345 is 46-fold higher than that of OV2964, but the abundance of mRNA is only 14 times. The rate of the abundance of mRNA and copis of DNA can be suggest the transcript effect as similarly single copy transcript numbers and we can find that the transcript effect of OV2964 is 3-fold higher than OV1345 really. It suggests that the first intron of OV gene has a cis-regulation function to OV gene transcription. Although no green fluorescence signal was enough to detect in two lentiviral vectors with OV gene, the results of selection of antibiotic could also imply that the immortalized oviduct epithelia have some physiological characteristic of natural oviduct epithelia without of CEF cells.