Preparation Of In Vitro Reassembled Human Follicle-stimulating Hormone And Construction Of Transgenic Mouse Model

Follicle stimulating hormone(FSH)is a glycoprotein hormone which composed of FSHα and FSHβ subunits through non-covalent bonds.The dissociated FSHα and FSHβsubunits do not possess biological activity unless they are assembled correctly.In adult females,the main role of FSH is to stimulate the development of follicles,ovulation and growth of the endometrium.In men,FSH stimulates the production of sperm and development of secondary spermatocytes,and promotes sperm development and maturity through the synergistic effect of LH and androgens.Clinically,FSH is mainly used for the preparation of test-tube babies and the treatment of infertility.The FSH products currently on the market mainly include urine-derived human FSH(uh FSH)and genetically engineered recombinant human FSH(rh FSH).Howerver,uh FSH has the disadvantages of uneven source and contamination by impurities.The rh FSH is considered to have a uniform source and good biological activity.The commercialized rh FSH products are mainly produced by mammalian cells,such as Puregon and Gonal-F,which are secreted by Chinese hamster ovary cells(CHO)cells.It is worth noting that the rh FSH proteins produced by cells are relatively low in yield and expensive.With the development of transgenic technology,the animal mammary gland bioreactors are known as a promising method in preparation of exogenous recombinant proteins.The exogenous proteins prepared by animal mammary gland bioreactors gain complete post-translational modification and have similar structure when compared with the natural protein.However,there are no reports on preparation of rh FSH through transgenic large animal mammary gland bioreactors which is due to the excessive accumulation of biologically active FSH in animal mammary glands might cause animal diseases and even tumors.Therefore,it is still needs further research and exploration to prepare rh FSH through transgenic large animals mammary glands while avoiding the risk of tumors in transgenic animals.The main results of this study are as follows:(1)Expression of human FSHα and FSHβ subunits The pIRES-Hyg3-FSHα and p IRES-Neo-FSHβ/His eukaryotic expression vectors were constructed and transfected into CHO to express single human FSHα and FSHβsubunit.The cell lines CHO-FSHα and CHO-FSHβ with stable expression of human FSHα and FSHβ subunit were obtained.The p Ad-FSHα and p Ad-FSHβ/His adenovirus expression vectors were constructed and transfected into HEK 293 A cells to obtain recombinant adenovirus Ad-FSHα and Ad-FSHβ.The bovine mammary epithelial cells,goat mammary epithelial cells and goat mammary galnds were infected by Ad-FSHα and Ad-FSHβ adenoviruses to harvest FSHα and FSHβ subunit proteins.The FSHα and FSHβsubunits were digested by PNGase F to detected the glycosylation.Results showed that the FSHα and FSHβ subunits from bovine mammary epithelial cells,goat mammary epithelial cells and goat mammary glands possessed N-terminal glycosylation modification.(2)Establishment of in vitro reassembly method of human FSHα and FSHβ subunits The FSHα and FSHβ subunits expressed by CHO cells were purified and reassembled in vitro at different concentrations at 4°C with a molar ratio of 1:1.Results of His tag pull-down analysis showed that the FSHα and FSHβ subunits expressed by CHO cells could be reassembled into a protein in vitro.In addition,results of ELISA assay indicated that the in vitro reassembly efficiency of FSHα and FSHβ subunits increased with the increase of concentration of FSHα and FSHβ subunits.Based on established method,the FSHα and FSHβ subunits expressed by bovine mammary epithelial cells,GMECs and goat mammary glands could also reassembled in vitro successfully,which further proved the feasibility of in vitro reassembly of FSHα and FSHβ subunits.(3)Study of biological activity recovery technology and biological activity on reassembled rh FSH The in vitro stability environmental conditions(temperature,concentration,p H value)of reassembled rh FSH were optimized in this study.Results demonstrated that the reassembled rh FSH showed better stability at 4°C,p H7.4 and high concentrations.The reassembled rh FSH from CHO,bovine mammary epithelial cells,goat mammary epithelial cells and goat mammary glands were determined bioactivity.The results showed that the reassembled rh FSH could bind to the FSHR receptor stably expressed on the HEK293/SNAP-FSHR cell membrane to stimulate the endocytosis of FSHR and promoted the production of cyclic adenosine monophosphate(c AMP)in cells(P <0.01).In addition,theof reassembled rh FSH(6 pmol,8 ×)could significantly promote the ovarian weight gains(P < 0.01)and stimulate follicle maturation and endometrial growth in rats.Furthermore,10 pmol of reassembled rh FSH also significantly increased the number of ovulation in mice(P < 0.01).In summary,the technology of reassembled rh FSH biological activity recovery was established successfully and could regained biological activity of reassembled rh FSH from CHO cells,bovine mammary epithelial cells,GMECs and goat mammary glands.(4)Establishment of gene targeting system for human FSHα and FSHβ genes integration in goat BLG locus based on CRISPR/Cas9 technology Two gene targeting vectors p Cas9-sg1,p Cas9-sg2 and the donor vectors p GHA-FSHα,p GHA were constructed for targeted integration of the FSHα and FSHβ genes into the second exon region of the goat BLG locus using CRISPR/Cas9 technology.The p Cas9-sg2 vector was selected out for knock out in BLG locus of GMECs cells.After the p GHA-FSHα,p GHA-FSHβ/His and p Cas9-sg2 were respectively co-transfected into GMECs cells,2 strains of GMECs positive cell clones stably expressing FSHα-G and FSHβ-G proteins were screened out.After analysis of glycosylation and saliva acidification,the FSHα-G and FSHβ-G expressed by GMECs cells were found to have N-terminal glycosylation modification and sialylation.Similarly,the His tag pull-down results showed that these two subunits could be successfully reassembled into a complete rh FSH-G protein in vitro.Bioactivity analyses indicated that the rh FSH-G could bind to its receptor FSHR and stimulate cells to produce c AMP in vitro.Furthermore,the rh FSH-G also significantly promoted ovulation in mice and weight gain of ovaries in rats with a dose-dependent manner.Based on the established CRISPR/Cas9 system,the FSHα and FSHβ genes were targeted inserted into goat fetal fibroblasts to obtain nuclear donor cells for preparation of transgenic goats.In summary,this study established a CRISPR/Cas9 system for targeted insertion of FSHα and FSHβ genes in BLG locus and obtained the goat fetal fibroblasts with FSHα and FSHβ genes as nuclear donor cells for preparation of transgenic goats,which provided the foundation for the preparation of transgenic goats in the future.(5)Preparation and function study of transgenic mice with human FSHα and FSHβ genes The pBC1-FSHα and p BC1-FSHβ/His mammary gland-specific expression vectors were constructed and injected into mouse fertilized eggs by microinjection to prepare transgenic mice.After PCR identification,one transgenic female mouse with FSHα gene insertion and two transgenic female mice with FSHβ gene insertion were obtained.The Western blotting and ELISA were used to detected the expression of FSHα and FSHβsubunits in transgenic mice milk.Results showed that the FSHα and FSHβ subunits were expressed in the milk of transgenic mice,respectively.Furthermore,immunohistochemical analyses indicated that the FSHα and FSHβ subunits were significantly expressed on the inner wall of the mammary glands in transgenic female mice.The FSHα and FSHβsubunits from mice milk were reassembled in vitro and a series of experiments were applied to determined the bioactivity of reassembled rh FSH from mice milk.The results showed that the reassembled rh FSH gained bioactivity in vitro and in vivo.Importantly,the morphology of mammary gland and ovary of transgenic mice were not shown significantly different from that of wild-type mice.In conclusion,this study successfully prepared transgenic mice expressing FSHα and FSHβ subunits in the mammary glands,and further histomorphological analyses confirmed that the mammary gland and ovarian tissues of transgenic mice were not affected by the genetic modification.In summary,this study innovatively established the in vitro reassembly method of FSHα and FSHβ subunit proteins.In addition,by establishing the in vitro biological activity recovery technology of reassembled rh FSH,the biological activity of reassembled rh FSH prepared from CHO cells,bovine mammary epithelial cells,GMECs and goat mammary glands was regained in vitro.Furthermore,the CRISPR/Cas9 technology was used to establish system for the targeted integration of FSHα and FSHβ genes in BLG locus of goat and goat fetal fibroblasts transfected with FSHα and FSHβ target genes were obtained.Finally,the transgenic mice were prepared to prove the feasibility of reassembly of FSHα and FSHβ subunits from milk of transgenic mouse models and determine the influences of genetic modification on mouse breast and ovarian tissues.This study provided scientific basis for the future preparation of transgenic large animal mammary gland bioreactors to produce reassembled rhFSH.