Generation Of Transgenic Mice Model Expressing Human IFNα-2b In Mammary Gland

Interferon （IFN） are cytokines secreted by human body in response to the infections of viruses and other kinds of pathogens, which could defense against viruses invasion, besides, they also possess other important functions such as anti-proliferate, immune-modulating and cell apoptosis control, it is one of the most efficacious medicaments in curing of virosis and cancers. Based on the difference of biological properties, amino acid composition and the types of receptor, IFN were divided into several sub-types. IFNa-2b is one of the sub-types which broadly used in clinic in treating of hepatitis B and several kinds of cancers. IFNa-2b gene is located in9p21region of human chromosome9, encodes165amino acids. Mature IFNa-2b protein is a kind of glycoprotein which contains5a-helixes and2disulfide bonds; the molecular weight is about19.6kD.Hepatitis B is a global high incidence of infectious disease; an estimated350million people in the word are chronically infected, about6%of the global population. In China, there are about130million people infected, about10%of the total population. As an effective anti-virus medicine, IFNa-2b is in great demand. But, presently, all recombinant IFNa-2b used in clinic is synthesized by bacterial systems, thus lacks the essential right post-translational modification and spatially fold, thus the recombinant proteins were censured for their low biological activity, short blood half-life, and even trigger the development of antibodies in patient’s body. Therefore, transgenic mammary gland bioreactors have great prospects in producing large scale, full post-translational modified and high biological activity recombinant IFNa-2b. The objective of this study is to generate IFNa-2b transgenic mice mammary gland bioreactor models and laid technical and theoretic foundation for the generation of transgenic water buffalo mammary gland bioreactors.Jersey cow P-casein gene signal peptide containing promoter （5244bp,3912bp） and no signal peptide containing promoter （5165bp,3855bp）,1166bp ployA sequence, Holstein cow (3-casein gene signal peptide containing promoter （5219bp,3887bp） and no signal peptide containing promoter （5160bp,3830bp）,1166bp ployA sequence were successfully cloned,8promoter assay vectors were constructed with the8different cloned promoters （pEGFP-JS5.2BCNpsig, pEGFP-JS5.2BCNpnosig, pEGFP-JS3.8BCNpsig, pEGFP-JS3.8BCNpnosig, pEGFP-HST5.2BCNpsig pEGFP-HST5.2BCNpnosig, pEGFP-HST3.8BCNpsig pEGFP-HST3.8BCNpnosig.）, After transfected into human breast cancer cell line Bcap-37cells, with the stimulation of hormone, EGFP reporter gene were successfully expressed. These results showed that the8cloned promoters possess the promote ability. Then, IFNa-2b gene fused with6×His tag and screening gene cassette EGFP-Neo were also obtained. Finally,8mammary gland specific expression vectors were constructed （pJS5.2BCNsig-IFN-JSpA-EGFP-Neo,pJS5.2BCNnosig-IFN-JSpA-EGFP-Ne o, pJS3.8BCNsig-IFN-JSpA-EGFP-Neo, pJS3.8BCNnosig-IFN-JSpA-EGFP-Neo,pHST5.2BCNsig-IFN-HSTpA-EGFP-Neo, pHST5.2BCNnosig-IFN-HSTpA-EGFP-Neo,pHST3.8BCNsig-IFN-HSTpA-E GFP-Neo,pHST3.8BCNnosig-IFN-HSTpA-EGFP-Neo）. After transfected into human breast cancer cell line Bcap-37cell,9stable transfected cell clones were screened out. Quantitative Real Time-PCR, western blot and ELISA were performed in analyzing these9transgenic cell lines. Results showed that transgenic cell line transfected with pJS5.2BCNsig-IFN-JSpA-EGFP-Neo expressed the highest IFNa-2b level in these nine, and recombinant IFNa-2b possessed biological activity. So, this vector is suitable for transgenic animal’s generation.Neo gene in the candidate vector pJS5.2BCNsig-IFN-JSpA-EGFP-Neo was omitted, and transgenic mice models were generated by pronuclear microinjection. Transgenic mice were screened out by PCR and Southern blot, transgenic copy number, integration sites and transgene methylation statuses in different generations mice were also analyzed. Results showed that there were approximated3-6copies in these3founder transgenic mice. And7different integration sites located in4chromosomes were detected:one site in chromosome5,5q, region of NT₁66301.1; two sites in chromosome6,6p, region of NW₀01030811.1and NT₀39353.7; two sites in chromosome9,9p, region of NW₀01030922.1and NT₀39482.7; two sites in chromosome17,17p, region of NT₀39649.7and NT₁66301.1respectively. Base pair composition in the flanking arm of integration sites were AT rich. According to the propagation of transgenic mice, methylation statuses of CMV promoter increased generation by generation, from79.3%of founder mice,83%of Fl generation,85.7%of F2generation,93%of F3generation, to95.7%of F4generation. RT-PCR, Western blot, ELISA and Immunohistochemistry were also employed in analyzing the expression of IFNa-2b in transgenic mice’s mammary gland and milk. Results showed that IFNa-2b mRNA was successfully expressed in transgenic mice mammary gland during lactation, but not expressed in other tissues. These results reflected that transgene cassettes constructed in this study possess the property of mammary gland specific, Recombinant rIFNa-2b fused with His tag was confirmed by Western blot in milk samples, and the concentration of recombinant rIFNa-2b in milk was determined to be30μg/L by ELISA analysis. The expression of rIFNa-2b in transgenic mice mammary gland was also confirmed by immunohistochemistry.With the object of establishing a biological activity assay system for recombinant hIFNa-2b, hMxA gene promoter contains3ISRE elements was isolated. After promote efficiency assayed, expression vector pGL3-Mxp3was constructed. The same amounts of pGL3-Mxp3and pRL-TK vectors were co-transfected into WISH cells, and were treated with standard IFNa-2b samples, standard curve and regression equations between RLU value and IFNa-2b bioactive units were calculated out. Then bioactive units of recombinant IFNa-2b were assayed by WISH cells treated with transgenic mice milk, the biological activity was about255.85IU, and the specific activity is approximated2.8X10’IU/mg. The ability of recombinant human IFNa-2b to stimulate IFN-inducible genes’expressions was also assayed by QRT-PCR. Eight candidate genes were chosen, including MxA, OAS, PKR, Caspasel, p21, MAPK, MHC I and pD-1. Results showed that MxA, OAS, PKR and Caspasel were significantly increased in WISH cells treated by transgenic milk. Expression levels were:150folds （MxA）;10.72folds （OAS）;6.4folds （PKR） and3.5（Caspasel） folds increased respectively compared to that of the no-transgenic milk. But the expression level of p21, MAPK, MHC I and pD-1were not changed obviously. These, results indicate that the recombinant IFNa-2b expressed in transgenic mice mammary gland has full biological activity.