| With the development of transgenic animal technology, the research of chicken oviduct bioreactor has been one of the foci of transgenic studies. In this research, with the chicken ovalbumin 5'-flanking regulatory region (OV) and porcine interferon-βgene (INFβ) being successfully cloned, the recombined retroviral expression vector pLN-OV-INF was constructed . The recombined vector pLN-OV-INF and the expressive vector pVSV of retroviral env gene were co-transfected to the package cell GP2293 to transiently produce replication-defective progeny retrovirus. Then the retroviral stock was microinjected into the blastoderm to infect the blastoderm cells to produce transgenic chickens.At first, the genomes of chicken oviduct tissue and porcine ear tissue were extracted. The specific primers were used to amplify OV and INFβ. After gel electrophoresis, they were cloned into pMD18-T vectors (pMD-OV and pMD-INF) and were sequenced. The results showed that the INFβwas consistent with published sequence and though the OV had 5 mutations it can be used to promote exogenous gene expression in the chicken oviduct.The construction of recombined retroviral expression vector pLN-OV-INF had two steps. Firstly, the segment of INFβand the linear vector pMD-OV were got by cutting the pMD-OV and pMD-INF with the BamHI and MluI and were linked together to get recombined vector pMD-OV-INF. So INFβgene was linked at the downstream of OV. Secondly, the connecter of OV and INFβand the linear vector pLNHX deleted Phsp70 promotor were got by cutting pMD-OV-INF with BamHI and SalI and cutting pLNHX with XhoI和BglII. Therefore the connecter of OV and INFβgene was cloned into pLNHX to get recombined vector pLNHX-OV-INF. The recombined vectors were detected by the ways of PCR and digestion. And the results indicated the recombined vectors were constructed successfully.The recombined vector pLN-OV-INF and the VSV-G expressive vector pVSV were co-transfected to the package cell GP2293 to produce replication-defective progeny retrovirus by the way of transient transfection. Because the way of transient transfectiondoes not depend on the stable integration, the retrovirus stock was collected within the time of 24h to 72h after co-transfection. In this study, the retrovirus stock were collected every 24h or only once at 48h after co-transfection. The viral titer determination illuminated that lessening the collect times had not much role to increase the viral titer because that the half time of retrovirus is rather short at 37℃ and the VSV-G pseudotyped retrovirus can be concentrated by ultracentrifugation without loss of virus infectivity.About 5ul virus stock supplemented with polybrene (8ug/ml) was microinjected into the blastoderms of unincubated eggs. The injected eggs were transferred to the surrogate eggshellsⅠ,sealed with paraffin film and allowed to hatch at 38℃,50％ humidity in a automated incubator. Three days later, the eggs were transferred to the surrogate eggshellsⅡand were turned over by hands until the 16th of hatching. From the 18th , some little holes were made to help the embryos to breath. In this study, 13 chickens were hatched and 10 were still live.From the third day of hatching, 92 died embryos were selected to extract DNA. The 3-5th embryos were 27 and were extracted DNA in whole; the 6-9th embryos were 33 and were cut in two to extract DNA; 10-21st embryos were 22 and were dissected and their brains eyes hearts sexual glands and muscles were extracted DNA. The blood and the feather sacs of the10 live chickens were got to extract DNA. The samples of the died embryos and the live chickens in all were 223.PCR was used to detect whether there was porcine INF-βgene existed in the 223 DNA samples. In order to improve veracity and reduce false positive rate, positive and negative controls were made in every detection. Of the 223 DNA samples, there were 54 positive samples, and the positive rate was 24.2%, of which the died chicken embryos of 3-5th ,6-9th ,10-21st and live chicken w...
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