Production Of Goat Transgenic Embryos For Mammary Gland-specific Expression Human BPI

Human bactericidal permeability increasing protein（hBPI）is a kind of cationicprotein, which can act against gram-negative bacteria and neutralize endotoxins in humanpolymorphonuclear nertrophils. Generally, hBPI is used to treat certain kind of humaninfectious disease caused by gram-negative bacteria, sepsis and septic shock and otherdiseases. Additionally, hBPI also has neutrophil-mediated phagocytosis related opsonicfunction, fungistasis, anti-protozoan parasite, and inhibition of angiogensis, and otherbiological functions. Due to it holds great promises in human clinics, hBPI is also called akind of "super antibiotics".However, the traditional ways, such as column chromatography and chemicalsynthesis, to extract or produce this natural antimicrobial peptide hBPI, are challanging,complex and too expensive, which in turn usually limit the wide popularization andapplication of hBPI. Recently, genetic engineering offers the world a revolutional stratageto produce recombinant phamarceutical proteins in large scale, similar postmodifications tonatural proteins in mammary gland by producing transgenic mammary gland bioreactor. Tomake the gene of interest specifically and highly expressed in mammary tissues is one ofthe key upstream steps in making a transgenic mammary bioreactor. Hence, it is crucial tovalidate the structure integrity and function of the mammary gland-specific expressionvector immediately after construction, in order to avoid unnecessary work, save time,money and energy, and improve efficiency. Therefore, the objective of the present studywas to construct mammary gland-specific expression vector, validate the construc by cellculture, transgenic mouse model, and production of goat embryos by transgenic cloning,in order to lay a foundation for future making of transgenic mammary gland bioreactor toproduce hBPI in large scale to satisfy clinical needs.The detai expriments were as follows:(1) Tissues from mammary gland of a Huanghuai White goat, at60d of gestation but notin lactation period, were collected, and the explants were seeding on non collagen coatedpetri dishes, drying for10h before adding medium DMEM/F12supplemented only withFBS, insulin-transferrin-selenium and epidermal growth factor. Enzyme differentialdigestion, high density cultivation and continuous passaging were performed to purify goatmammary epithelial cell lines, whose morphology, specific antigen expression, growthcurve, chromosome number, milk lipid synthesis, and in vitro culture viability were examined. Our results show that the lactating breast tissue in simple culture conditions canalso give rise to lactation mammary epithelial cell line.(2) Sequence of the mammary gland-specific expression vector was design as XhoI+Kozak sequence+Bovine β-casein signal peptide+coding sequence of hBPI+Xho I.Then the above sequence were synthesized and cloned to EcoR V site of pUC-57, andnamed as pUC57-hBPI. This was subcloned into pBC1-Loxp-Neo-Loxp-pBD plasmid.Bacterium fluid PCR was performed to quickly indentify postive clones, and then t hePCR positive colonies were enzymatic cut furtherly followed by been sequenced. Allthe results indicate that the mammary gland-specific expression vectorpBC1-Loxp-Neo-Loxp-hBPI we obtained is structurally correct.(3) C127, a commercial mouse mammary tumor cell line, was transfected bymixing the pBC1-Loxp-Neo-Loxp-hBPI vector and Lipofectomine2000TM. Putativecolonies hopefully transgenic were obtained after screening by G-418for no less than7days. All clones that alive were mixed together, and then cultured in hormone inductionculture media. RT-PCR results showed that transcription of hBPI is happening althoughat low level; Western blot test showed that the construct containing hBPI can betranslated; Lymphocyte transformation test showed that the recombinant hBPI canstimulate the proliferation of lymphocytes and the neutralization of endotoxin (LPS)biological activity, which means the expressed recombinant hBPI is functional. Theresults demonstrated that the structure and function of constructed vector is feasible formaking mammary gland bioreactor producing human BPI protein.(4) Founder transgenic mice expressing hBPI were generated by DNAmicroinection. Milk protein and tail samples were collected in order to examine if themouse is transgenic and could express active hBPI. The results indicated that7foundermouse(5、2) of of hBPI transgenesis were obtained. Unfortunately, due to poorhousing environment, health conditions and other factors, all founder mice have died.Further studies are still needed to explore if our construct can express at high level andhighly active.(5) Guanzhong dairy goat fibrablast were, as host cells, were transfected by themixture of pBC1-Loxp-Neo-Loxp-hBPI and Lipofectamine2000TM. Transfected cellswere screened by culturing in Medium containing500μg/mL G418for14-16days.Large numbers of G418-resistant clones were obtained. Colonies were picked forexpanded culture. PCR of picked colonies was performed, and the results showed thatthe exogenous genes is integrated into the G418resistant cell genome. Four transgenic cell lines, GZ-GFC-1(♀),GZ-GFC-2(♀),GZ-GFC-3(♂) and GZ-GFC-4(♂) expressinghBPI were achieved.(6) The transgenic cell lines GZ-GFC-1(♀), GZ-GFC-2(♀), GZ-GFC-3(♂), andGZ-GFC-4(♂) were used as donor cells to produce transgenic somatic cloned embryos bySCNT and Handmade Cloning (HMC). Donors from All four transgenic cell lines wereable to support preimplantational embryonic development to the blastocyst stage.(7) By using HMC, we produced125cloned embryos from the donors of GZ-GFC-2(?) and GZ-GFC-3(♂) cell lines. Some blastocyst were tested positive by PCR. Some ofthe HMC embryos were transferred to15recepient Huang Huai white goat of natural heat,and sadly, all surrogate mothers returned to estrus one month after transfer. The underlyingmechanism of poor in vivo development of these hBPI transgenic embryos requires furtherstudies.In conclusion, a mammary gland-specific expression vector of hBPI is successfullyconstructed, and the structure integrity and function is validated by a simple, efficient cellculture platform, and cloned embryos expressing hBPI were also successfully produced byan easy leaning, stable, and simple somatic cloning method HMC. Moreover, A Huanghuaigoat mammary epithelial cell line is established from non-lactating goat. In all, our resultlay a solid foundation for future study on making transgenic goat expressing hBPI.