Culture Of Chicken Stage X Blastoderm Cells And Production Of Chicken Oviduct Bioreactor

With the development of transgenic animal technology, the research of chicken oviduct bioreactor had been one of the foci of transgenic study. In spite of many efforts, the production efficiency of transgenic birds is still substantially poorer. To develop an approach to producing efficiently transgenic chickens as bioreactors was a new question .It is a novel strategy for introducing an interest gene under the control of an endogenous promoter by gene gartgeting. In fact ,many genetic manipulations require cells which are able to contribute to both somatic tissues and the germline.These cells should be maintained in culture over a period of time in vitro while the cells were screened for successful transfection, intergration of the transgene vector. In present study, the X stage blastdem cells were cultured and labeled , followed by a method of producing an integrated transgene in an avian cell and screening for nuclei acid integration in a cellular genome ,and vectors used for transfected blastodermal cells to produce transgenic avian as bioreactor were constructed . The results were as follows:1. The putative U3-R sequence regions of embryonic normal stem cell gene 2 (cENS2)of Shouguang chicken was cloned and successfully sequenced. Based on sequence analysis and alignments of nucleotides sequence ,we found an important elements( B region) which exhibits a strong enhancer activity in CES cells. The CMV promoter was cut off from pEGFP-N1, and the putative U3-R fragment was inserted just upstream of GFP gene. Results of restriction digestion and DNA sequencing showed that the vector pEGFP-cENS2-Promoter for expressing in CES cell was successfully constructed.The CEF cell and blastodem cells were transfected by the vector ,we found the U3R promoter activity was restricted to the blasderm cells2. The monolayers of blastodermal cells drived from area pellucida and area opaca were respectively transfected with the vector pEGFP-cENS2-Promoter ,the promoter activity in cells of pellucida area was stonger compared with that of opaca area,implying that CES cell mainly distributed in the pellucida area of X stage embryo.3. The monolayers of blastodermal cells were transfected with pEGFP-cENS2-Promoter and pEGFP-N respectively, the results suggest that part of blastodermal cells cultured for a week in vitro still have the CES activities.After selection by G418, we had not got the genetically modified blastodermal cells were not acquired.4. We cultured chicken blastodermal cells on a mouse feeder layer in presence of BRL-3A conditioned complete media which was composed of DMEM (high glucose) supplemented with 2% chicken serum, 0.1 mM β -mercaptoethanol, 2mM L-glutamine, 10mM HEPES(PH7.6), 20ng/ml conalbumin, 100U/ml penicillin, 100 μ g/ml streptomycin, 10ng/ml gentamycin, fetal bovine serum and cytokines. The avian cells produced express an CES phenotype which had the character of AKP and PAS positive and were cultured for a short period of time.5. The dispersed blastodermal cell were transfected with the vector pEGFP-cENS2-Promoter,then these blastodermal cells were plated directly onto SNL feeder cells and cultured with BRL-3A conditioned complete CES medium. Cells were seclected by G418 of stable BCs clones which confirms the success of this transfection procedure, allowing us to use modified cells for generating transgenic chickens.6. To generate a recombinant vector pLNHX-OV-IFN-IRES-EGFP(±) ,the HSP70 promoter ofpLNHX, a replication deficient vector, was replaced with an expression cassette which include an ovalbumin (OV) 5' flanking regulatory region, a swine interferon beta gene, the internal ribosome entry site(IRES) element and the reporter gene GFP.7. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 polyethlenimine nitrogen per DNA phosphate .The purified recombinant vector was mixed with polyethyleneimine and injected into the wing vein of laying-egg hens. By RT-PCR analysis, we found that the transfected gene was specifically transcribed in the oviduct, which suggested the vector could used to produce transgenic chicken as bioreactor.8. The vector pLNHX-OV-IFN-IRES-EGFP(+) .and pVSV-G were co-transfected with the GP2293packaging cell , we got titers of 10~5 and injected into the subgerminal cavity of the blastoderm. The injected eggs were sealed with eggshells and paraffin and allowed to hatch at 38℃, 50% humidity in a automated incubator. In this method, two chickens were hatched .9. The 7.8 kb chicken OV gene genomic DNA sequence for homologous arms had been cloned , then the m2/Loxp71-EGFP-Loxp66-IRES was constructed and subcloned between OV 5' UTR and CDS, the thymidine kinase (tk) as the negative selection marker gene, was just outside the arm. Chicken OV gene targeting vector pSSC-m2/71EGFP661RES-OV7.8 containing a Cre "exchange cassette" was constructed, which would be used for developing chicken oviduct bioreactors.