Production Of Dairy Goat Mammary Gland Bio-Reacotr For Human Lysosomal Beta-Glucosidase Using Transgenic Cloning Strategy

Human lysosomal acidβ-glucosidase(G1cCerase) is responsible for the catabolism of glucosylceramide. Deficiency of GlcCerase activity results in the progressive accumulation of glucosylceramide, and it ultimately leads to clinical manifestations of anemia, hepatosplenomegaly, bonelesions, and in more severe cases, central nervous system impairment, which called Gaucher disease (GD) in clinical. GD is now treated mainly by administrating the missing enzyme (enzyme replacement therapy, ERT). Human GlcCerase is a difficult enzyme to be obtained, at present, CHO cells and transgenic plants systems are used for the production of recombinant G1cCerase, but which remian limited by their high cost in CHO cells and difficult purification and hypo-glycosylation in transgenic plants. Transgenic animal bioreactor offers particularly attractive possibilities to prepare recombinant G1cCerase, with the advantages of low cost production as well as high quality proteins. However, pronuclear microinjection have been used for more than two decades to produce transgenic animal, the low efficiency and high cost has been the main barrier for transgenic animal production. Nuclear transfer using transgenic donor cells has provided an alternative, efficient technique for the production of transgenic animals expressing a protein of interest. Therefore, we selected the human GlcCerase as the targeted object. Firstly, GlcCerase cDNA sequence was cloned and the biological activity of the protein was studied. Secondly, mammary-gland specific expression vector of GlcCerase cDNA was further constructed and the effectiveness of this vector was verified in in vitro mammary epithelial cells. Thridly, this mammary-gland expression vector was transfected into dairy goat fetal fibroblast cells, and stable transgenic cells integrated with G1cCerase gene were obtained by selection. Finally, the transgenic goat cloned embryos were produced by SCNT, in order to obtain transgenic goat mammary gland bioreactor expressing G1cCerase.The study was divided into six parts, the first and second parts focused on the cloning of G1cCerase gene, construction of GlcCerase expression vector and its expression in vitro, which provided basis for the expression of this vector in the mammary gland; the third parts and fourth parts were to optimize the transfection system of dairy goat fetal fibroblasts using liposome, and obtain donor cell lines stablely integrated with humanβ-glucosidase gene; the fifth part consists of using the transgenic cells as donor cells of nuclear transfer and evaluating the developmental ability of goat cloned embryos derived from these transgenic cells in vitro and in vivo. The sixth part was to identify the effect of GlcCerase gene transfection into dairy goat somatic cells in terms of cell cycle distribution, chromosome abnormality, incidence of apoptosis, and the relative abundance of gene expression. The main results were as follows:1. We amplified human GlcCerase gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T vector, the gene homology was 99% comparable to that of the reported human GlcCerase cDNA sequence in GeneBank, and one nucleotide difference was found, resulting in the change from aspartic acid to glycine. To test whether the cloned gene could encode the recombinant protein correctly, the GlcCerase gene was further subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-G1cCerase. After the recombinant plasmid were identified by restriction enzyme digestion, we transfected pEGFP-G1cCerase into COS7 cells by liposome, GlcCerase mRNA was expressed and the activity of GlcCerase was also detected in COS7 cells. These results indicated that the cloned human GlcCerase gene could encode proteins correctly and play a biological function, which could be used in the next step of mammary gland expression vector.2. The GlcCerase verified by the eukaryotic expression and the neomycin resistance gene (Neor) to permit selection of transformed cells were subcloned into pBCl vector containing goat beta-casein promoter respectively, then the constructed vector pBC1-GlcCerase-Neo was then proved by enzyme digestion and PCR amplification. In order to analyze the bioactivity of the vector, pBC1-G1cCerase-Neo was transfected into mouse mammary epithelial cell line HC-11. Positive single clone cells were selected with G418 and by PCR. The transgenic cells were cultured in induction medium containing RPMI-1640 medium with prolactin, insulin and hydrocortisone, which could induce recombinant human GlcCerase expression. RT-PCR and Western-blotting analysis showed that goatβ-casein gene promoter could regulate the transcription and translation of human GlcCerase gene in mammary-gland epithelial cells.These results indicated that the constructed expression vector of pBC1-G1cCerase-Neo had biological function, and could be used for further establishing transgenic donor cells with human GlcCerase gene.3. In order to prepare donor cells for dairy goat transgenic cloning, goat fetal fibroblasts cells (gFFCs) were isolated by attaching tissue explants from a day 30 goat fetus and purified by trypsin. The gFFCs were examined by cell morphology, growth curve and karyotype of chromosome, sex-determined region Y gene (SRY) of the gFFCs was also identified, which indicated that it’s suitable for the need of transgenic clone. Important factors involved in cationic liposome mediated gene transfer were also evaluated through in vitro transfection of gFFCs:the concentration of DNA and liposome, the effect of transfection time on the efficiency of gFFCs to express a reporter gene (GFP). The results showed that gFFCs cultured in 24-well culture plates with 4.0μL liposome and 1.2μg plasmid DNA for 6 hours resulted in the highest transfection efficiency, which was 4.21%. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer.4. The dairy goat fetal fibroblasts were transfected with linearized plasmid pBC1-G1cCerase-Neo using liposome by the optimized procedure and selected with G418. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates. The expanded clones were identified by PCR, the results indicated that the transgene was stably integrated into the open region of the chromatin of G418 resistant fibroblast cells. And the transgene did not result in the abnormalities of cell growth and chromosome ploidy, the percentage of transgenic cells with normal chromosomal number (60) were similar (66.8±3.2%) to those in control cells (70.9±0.8%, P>0.05). The above results indicated that these transgenic cell clones may be competent as donor cells for creating a transgenic goat by SCNT.5. Goat ovaries were obtained from a local abattoir and the cumulus-oocyte complexes (COCs) were matured in vitro,63.3% COCs were matured. Following nuclear transfer, stable fibroblast cell lines and non-transfected cells as control synchronized in Go by fully confluency for two days were used as donor cells respectively, MⅡoocytes without nuclear and first polar bodies were used as receptor cells. These reconstructed embryos were fused for 20μs under 1.2 kv/cm voltage, and activated using ionomycin and 6-DMAP, then co-cultured with cumulus cells in SOFaa. Similar rates (P>0.05) of fusion (83.3 vs 77.8%), and developmental capability for 2-4 cells (89.1 vs 90.9%),8-16 cells (50.9 vs 53.2%) and morula/blastocyst rates (36.4 vs 38.9%) were found between these two groups. Moreover, a total of 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients with two goats showing pregnancy at day 40. Unfortunately, the pregnancies failed to maintain to term, and the two fetuses aborted.6. Our study attempted to indentify the effect of gene transfection of human G1cCerase gene into dairy goat somatic cells on the characteristics and the relative abundance of gene expression, pBC1-G1cCerase-Neo plasmid were transfected into goat fetal-derived fibroblast cells (FFC), mammary epithelial cells (MEC), and adult ear skin-derived fibroblast cells (AEFC) respectively, and then transgenic somatic cell lines integrated with G1cCerase genes were obtained.The results showed that lower percentage (P<0.05) of cells at G0/G1 in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by the flow cytometric analysis. Further, we also examined the expression of genes involved in imprinting (IGF2, IGF2R), apoptosis (Bax), stress (heat shock protein, Hsp70.1), cellular connections (CX43) and DNA methylation (DNMT1) in transgenic T-FFC. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared to non-transfected FFC.This is the first study evaluating the influence of foreign gene transfection on dairy goat somatic cells in terms of cell cycle distribution, chromosome abnormality, incidence of apoptosis, and the relative abundance of gene expression, for exploring the inherent mechanism of low efficiency in transgenic cloning, and promoting the programming of donor cells and further improving the efficiency of transgenic cloning.