Construction The Transgenic Chicken Technology Platform With The Lentvirual Vector

In 1980,the first transgenic mouse was produced by the DNA microinjection. Since then, the scientists have being try their best to produce the transgenic chickens. Because the transgenic chickens are essential for the basic research and biotechnology, so creating an efficient technology of producing transgenic chicken has being a hot research area.Though the foreign countries have produced the transgenic chickens, there is no transgenic chicken in China. In our research, we will focus on producing the transgenic chickens through the lentvirus vector with the EGFP gene as the Marker gene. We hope the foreign gene can be ingrated into the sperm genome and be passed to next generation, so that we can create an efficient system of producing transgenic chickens as soon as possible in China.The quality of the chicken embryo plays am important role in successfully producing the transgenic chickens. Through our research, we found that the chicken embryo of the Dawu Group was better than others.There are two ways to culture the chicken embryo after being modified, One is the windowing method, the other is to culture of chicken embryos in Surrogate Eggshells. In this research, we absorb the windowing method. After making the newly laid eggs disinfected, Then the eggs were positioned with their sides facing upwards for overnight at room temperature in order to fix the blastoderm position. After swabbing the shell with 70% alcohol, a 5 mm×5mm sized window was made in the equatorial plane of the eggshell using a fine drill, which was followed by removal of the small shell membrane inside the window with fine forceps and a surgical blade. Then some thin albumen are added, and the window is sealed with the paraffin and the paraffin film. Eggs were incubated with the narrow end down at 38.2℃/65% relative humidity and were turned 90°each hour from the first day to the fifth day. In the following 13days , the hatch conditions is 37.8℃/60% relative humidity and were turned 90°each hour. From the 19~th day, Eggs were then placed in hatching baskets and incubated at 37.2℃/70% relative humidity until hatch. The result indicated that hatchability of sealing with the paraffin was superior to the paraffin film.Following, we explored how the microinjection could affect the hatchability. About 2～2.5 microliters of DMEM was injected into the central part of the subgerminal cavity by using a microinjection pipette. We set 25 fertilized eggs, as a result, there were 9 hatched eggs. The hatchability is 36%。This hatchability could satisfy the need of producing the transgenic chicken.In this research, the transgenic chickens were produced with the lentiviral vectors. Now, this way is the most successful method to produce the transgenic chickens. The lentiviral vectors that we use are composed of the transfer vector, packaging vector and envelope vector. As for the transfer vector, the gene is the enhanced green ?uorescent protein gene as the Marker gene. The titre is 1×10~9 TU/mL.To develop the transgenic chickens expressing enhanced green ?uorescent protein, approximately 2～2.5ul of viral suspension was microinjected into the subgerminal cavity beneath the blastodermal embryo of newly laid eggs,the titre is about approximately 1×10~9 transducing units per millilitre (TU/ml).The injected embryo were cultured to hatch. In all we had injected 110 embryos successfully, as a consequence, we got 32 hatched Go chickens. When the chickens were older, we extract the DNA genome from the blood of the vein of the chicken wing. Through the PCR screen of hatched male and female chickens, there were four chickens that contained the EGFP gene and the part vector sequence in their blood genome. There were 16 cockels in the 32 Go chickens ,when they were raised to sexual maturity, the genome DNA were extracted from semen samples. Similarly screened by PCR and sequencing, we confirmed that 6 cockels contained the EGFP gene and the part vector sequence in their semen genome.In all, our research has laid a solid basis for producing the transgenic chickens.