Construction Of Oviduct-specific Expression Vectors And Study On Transitory Expression System

In order to construct oviduct-specific expression vectors, we cut thesequence of 5'-flanking region of chiken ovalbumin promoter, whichcontains the 5'-f1anking sequence and the first extron and the sequenceencoding first l40 amino acids.In order to study the structure and function of chicken lysozyme gene,we cloned the sequences of 5'-flanking region of chiken lysozyme promoter(52lbp ) and its upstream sequence of hormone responsive element(44lbp ) by PCR .The former contains the start codon ATG and the firstpart extron and the whole signal peptide sequence, the later includes thesequence sites with which progesterone and glucocorticoid receptor bind.Matrix Attachment region (MARs) is a DNA fragment which can insertiqto a domain of untranslation region (UTR) and binds with proteinaceousmatrix then make chromatin form a chromatin loop, which is an activetranscription unite and can reduce position effect, in result of improving thetranscription efficiency. We cloned the MAR sequence of chicken globingene, finished the plasmid of pOVghMAR, which was transfected intoprimary chicken oviduct cell culture, but we have not detected the increaseof expression product. We find the reason is that only when the transgeneconstruct containing MARs integrated into the genome can it make a roleand it has little use in the transient expression system.The sequence of human growth hormone gene covers 1964bp,containing 5 extrons and 4 introns. Foreign protein(hGH )is synthesized andsdcretes into culture solution, avoiding cell-crushing .As a good reporterprotein it is easy to be detect. Above all, hGH has a high stability in mostmamma1 cells.Based on the vector pBluescript and PUCl9, with chicken ovalbuminpromoter, chicken lysozyme promoter (52lbp) and further sequence ofhormone responsive element (44lbp) and reporter gene hGH, constructedfour kinds oviduct-specific expression vectors -- pOVghMAR, POVFgh,pLYSgh and pELYSgh.Transfected fresh oviduct cells by Dosper-mediated method andelectroporation with above vectors, we detected the expression product inculture solution after incubation for 24 hours and reach the maximum 48hours and begin to drop after incubation fOr 72 hours. We foundelectroporation is better than Dosper-mediated method significantly.We injected four vectors into living chicken oviduct by operation andthen applied electroporation in injected region. After 48 hours, killed andcollected extraction of injected region, and detected foreign geneexpression. But because there is chicken growth hormone in chicken body,which has 70% homology with hGH, ground error will happen sometimes.Concludingly, we take some road in researching versatile expressionvector by construction oviduct-specific expression vectors, which is veryimportant to study transgene bioreactor. The establishment of cellincubation system and electroporation of living oviduct, offers a platform todetect the biology of new transgene constructs.