Construction Of Oviduct-specific Gene Targeting Vector And Establishment Of Immune Tolerance Model In Chicken

Considerable progress has been made in developing methods for the production of transgenic animals and specifically in the expression of therapeutic proteins in the mammary glands of cows, sheep and goats for over twenty years. Transgenic chicken is also expected to be an excellent bioreactor for the production of recombinant pharmaceutical proteins. This research is to establish the fundamental elements for the production of transgenic chicken and attempt to resolve the immune response on transgene espression by inducing immune tolerance in chickens.1. The construction of chicken oviduct cell specific targeting vectors and secretory fusion vector1.2kb ovalbumin 5' regulatory region and lysozyme signal peptide sequence was Cloned by PCR from the vector pOVALG A 3.0kb DNA fragment of ovalbumin gene including 1.2kb 5' regulatory region and its signal sequence was gained by restriction endonuClease from the vector pOVAG-2. The two DNA fragments are upstream homologous arms of two gene targeting vectors, respectively. Another 2.7kb DNA fragment of ovalbumin gene was cloned by PCR from the genome of chicken blood cells for using as downstream homologous arm. The targeting vectors, pOVALs-targeting and pOVA-targeting, were obtained by inserting upstream and down stream homologous arms into the vector pEGFP-N1 with addition to HSV-tk gene outside the all homologous arms, respectively. The cDNA sequence of human obese gene was cloned from the vector HOB and inserted into the multi Clone site of pEGFP-Nl, which made it possible to co-expression with GFP. The secretory fusion vector phLepGFP-secretion was constructed after lysozyme signal sequence added.2. Expression of the vector phLepGFP-secretion in eukaryotic cellsThe vector phLepGFP-secretion was transfected into the chicken embryo fibroblast, and green fluorescent light was observed in the cells. This indicated that transgene could be expressed successfully. After that, the vector was transfected into chicken bursal lymphoma cell line (DT40) to obtain the transgenic cell line with the presence of G418. Forty four of 52 samples were confirmed to be positive, which meant transgene were integrated into the genome of DT40 cells. The cell culture supernates were detected by ELISA to explore if human leptin existed. The result showed that human leptin was secreted to the cell culture supernates, and the concentration of leptin was up to 512pg/ml per 10~6 cells.3. Study of inducing immunological tolerance in chickenCasein, BSA and HSA were respectively inoculated into chicken embryos by embryonic blood vessel microinjection at early stages of embryonic development for inducing immunological tolerance. The hatched chicks were challenged with the same antigens using in embryonic experiments. The serum samples were collected at 2 months old, and anti-toleragen antibodies were detected by ELISA. The results showed tolerance could be observed in over 54% chickens exposed to antigens in embryo at 65-70 hours of incubation, while not in chickens exposed to antigens at 70-74 or 88-113 hours ofincubation.BSA and HSA were were respectively inoculated into chicken embryos by in ovo inoculation at embryonation days 3-7. The hatched chicks were treated the same as above description. The ELISA result showed tolerance could be induced when the first embryonic inoculation occurred at 5 or 6 day of incubation. The production of anti-toleragen antibodies were also suppressed post hatch when the first embryonic inoculation occurred at 7 day of incubation, however, shorter duration of tolerance.In conclusion, our study is useful for preparation of transgenic chicken and oviduct bioreactor.