Construction And Expression Of Oviduct-specific Expressional Vectors In Quails

A great progress has been got in the study of mammal bioreactor,scientists have established bioreactor of cows and sheep to produce exogenous protein.Because of the special reproductive and physiological model in birds,results in the stagnancy of bioreactor studying of birds,one major obstacle to make bird bioreactor is that the specific expressional vector which can control the expressing of exogenous gene was not constructed.The establishment of upstream vector is the first important element.The expression of exogenous gene in oviduct of quail must have the participating of tissue-special promoter.Ovalbumin constitutes more than half of the protein in the white of a laid egg,and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct,the 5'-flanking sequence of ovalbumin which used by many researchers is tissue-specific and induced by hormone.In order to solve the problem,utilize the advantage of bioreactor rely on birds,accelerate the progress in producing chimeric quail.Here,we use quail as a platform to study.Some aspects of the research are as following:1 The cloning of 5'-flanking sequence of ovalbumin in quailThree different sequences(OV1,OV2,ERE)of 5'-flanking of ovalbumin in quail were successfully amplified by PCR.The sequence of OV1 locates about 1.1kb in transcription start site of the 5'-flanking of ovalbumin of quail;The first intron,exon and parts of the second exon sequence which included in the sequence of OV2 locates about 2.Skb in transcription start site of the 5'-flanking of ovalbumin of quail;The sequence of ERE which is 675bp is the estrogen-responsive enhancer element in 5'-flanking of ovalbumin in quail.2 The construction of GFP expressed vector driven by 5'-flanking sequence of ovatbumin in quailThe expressed vectors of pGFP-OV1,pGFP-OV2,pGFP-OV-ERE were constructed by replacing the CMV IE promoter in pGFP-N2 with OV1,OV2,ERE.Regulatory sequences and parts of the exon/intron structure of the gene were used to generate the following constructs:the construct designated pGFP-OV2 contained the steroid-dependent regulatory element,the negative regulatory element,exonl,intronl,and the beginning of exon2(OV2,2.8 kb).The estrogen-responsive enhancer element(ERE)was cloned immediately 5'to the steroid-dependent regulatory element to generate construct pGFP-OV-ERE.3 The expression of three recombined vectors in primary oviduct epithelium and embryo fibroblasts cells(QEF)of quailIn order to test the validity of three recombined vectors we constructed,the primary oviduct epithelium and quail embryo fibroblasts cells(QEF)of quail were cultured as testing platform.The QEF and primary oviduct epithelium were infected with the three recombined vectors, It shown that neither of the three recombined vectors was expressed in QEF cells whereas all of the three recombination vectors was expressed in oviduct epithelium primary but the effect is different. When the cells were infected 48h,the number of GFP-Positive cells of the three recombined vectors achieving peak,the number of pGFP-OV-ERE was higher than pGFP-OV2(P＜0.05);pGFP-OV2 was significantly higher than pGFP-OV1(P＜0.01);pGFP-N2 was significantly higher than pGFP-OV1,pGFP-OV2 and pGFP-OV-ERE(P＜0.01).