Study On A New Method Of The Lentivirus Production And Development Of Transgenic Chicken Bioreactor

Over the past decades,the study on genetically modified chicken in unremitting efforts of scientists,has considerable progress.This field is widely concerned,not only because the chicken is an ideal animal model,but also the possibility of the transformation of genetically modified chicken into human medicinal protein biological reactor.More efficient and cheap than the traditional industrial reactor and mammalian cell bioreactor has become the consensus when evaluating bioreactor of avian oviduct.It also develops the study of transgenic chicken which has become the new trend of scientific research and pharmaceutical industry.Because of the particularity of chicken embryo development,we need in the transgenic chicken bioreactor preparation some special means which are different from in mammalian animal bioreactor.Currently,the use of lentiviral vector in preparation of transgenic chicken is regarded as the most reliable method.Because of the great defects of traditional packaging method of lentivirus and many other problems in the chick embryo injection and subsequent screening,the cases that successful preparation of transgenic chicken bioreactor are still less reported.In the first part of the experiment,we use polyethyleneimine(PEI)as a transfection agent,and polyethylene glycol 6000(PEG6000)as centrifuge medium in lentivirus production process,then determine concentrated lentiviral titer.Also several key factors that may affect the titer of lentivirus,such as initial N/P value,transfected plasmid DNA concentration,the choice of adjuvant are analyzed.Experimental results show that in 15-cm dish conditions:when the transfection of plasmid DNA total is 10.5 μg,N/P=20,PEI concentrated liquid storage pH=7.0-9.0,transfected 293T cells number is 0.5-2x 107,researchers can obtain good lentivirus.As a new transfection reagent,PEI can be used as a substitute for calcium phosphate and liposome for the production of high titer virus.In the second and third part,we use the a new optimized methods and a series of subsequent detection methods finally successfully obtain the transgenic chicken.Experimental results show that by the reformment of sealing mode,sealing membrane type,embryo puncture location,windows operating time,windows after the liquid seal processing and pollution prevention measures,we increased hatching rate to 55%.Among the hatched individuals,the proportion of transgenic chimeric individuals increased to 41.8%.After further screening of the 31 chimeric chickens,we finally obtained 4 male and 4 female reproductive gland chimeric transgenic chickens.In summary,this study demonstrates that PEI as a new transfection reagent can replace the calcium phosphate and liposome used in the lentivirus production.Compared with the two,PEI has a lower threshold and higher operation cost,so it has very high use value.We also introduced a complete method of production of transgenic chicken oviduct bioreactor.All of this maybe help researchers get transgenic chicken efficiently.