| Melatonin(MT),an indole neuroendocrine hormone, has many biologic fuctions. It primarily involved in circadian regulation of physiological, and its metabolic derivatives possess strong free radical scavenging properties. Meanwhile, it’s an antioxidant, can regulate the immune system. Laoshan dairy goat is a unique Chinese goat breed, with an excellent milk yield, will be a good model for mammary gland bioreactor for producing melatonin. It’s necessary to detect the expression of exogenous gene in the donor cells, before somatic cell nuclear transfer (SCNT). In this study we systematic discuss the mammary gland specific expression vectors of AANAT and HIOMT (two key synthase of melatonin biosynthesis)gene transfected mammary epithelial cells in the procedures, which can prepare for breeding the transgenic goat to produce melatonin fortified functional milk.In this study, we collected Laoshan goat mammary gland tissue for primary epithelial cell culture. AANAT and HIOMT gene was seperatly transfected into the purified primary cells in order to verify whether the cells can be uesd as genetically modified donor by testing the expression level of genes and the amout of melatonin synthesis. The results show as following:(1) We cloned AANAT and HIOMT gene from goat pineal. We amplified Neo fragment from plasmid pcDNA3.1-myc-his.This Neo fragment included an Not I restriction site in both upstream and downstream. Then, we used Not I enzyme to digest the Neo fragment and plasmid pBCl-AANAT/pBCl-HIOMT,and coneneted them to constructed two recombinant plasmid pBC1-AANAT-Neo/pBC1-HHIOMT-Neo,that can be resistant screened in eukaryotic cells. The results of bacterium PCR and enzyme digestion verification were postive, and the sequence of plasmids was correct.(2)We obtained the mammary gland tissue from the healthy dairy goat of differens sources,and cultivated the mammary epithelial cells by tissue mass inoculation method. Next, we purified the mammary epithelial cells by digesting with typsin to remove the fibroblasts. The mammary epithelial cells,closely arranged, had the typical featrues of epithelial cells as cobblestone-like cells. We could observe multi-core in the cells through high magnification microscope.In addition, The cells’growth curve similared to the "S", comply with the biology laws of cells. So the cells could be used to he following experiments.(3)We transfected the plasmid pBCl-AANAT-Neo and pBCl-HIMT-Neo into goat mammary epithelial cells by using lipofectamine 2000, respectively. We detected AANAT and HIOMT mRNA relative expression level in transfected cells and melatonin content in culture medium, that was collected when cells was cultured 48 h in induced culture medium.And five hydroxytryptamine(5-HT), a melatonin synthesis precursor, was added in the induced culture medium.The results showed both transfected AANAT and HIOMT gene in mammary epithelial cells have high expression of the corresponding gene, at the same time, the AANAT and HIOMT mRNA relative expression levels were significantly higher than those in control group (p<0.01); AANAT gene transfected mammary epithelial cells cultured in liquid with five hydroxytryptamine (5-HT) and the concentration of MT in culture solution was significantly higher than that in the control group (p<0.01). It indicated that the transfection system could successfully transfer AANAT and HIOMT gene into Laoshan dairy goat mammary gland epithelial cells. In the presence of melatonin synthesis precursor 5-HT, melatonin synthesis of experimental group was significantly higher than that in control group (45.31±2.32 ng/mL vs 36.14±1.82 ng/mL,p<0.05)In summary, the recombinant plasmid pBCl-AANAT-Neo/pBCl-HIOMT-Neo, Laoshan dairy goat mammary gland epithelial cells and transfection system in this study can be used for the upstream experimental for mammary gland bioreactor. |
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