Study On Mapping Of Salmon Calcitionin In The Goat

Transgenic animal is an organism whose genetic material has been altered using genetic engineering techniques. With the rapid development of biotechnology, the way through the preparation of transgenic animals to produce protein drugs is becoming current hot research. Salmon calcitonin is an effective drug for treatment of osteoporosis, bone pain and some others, which has a wide range of applications in many countries and regions, and its output still can not meet demand. However, by manufacturing transgenic goats, its mammary gland can produce a large number of salmon calcitonin. This has good cost-effectiveness and market prospects. In this paper, we studied genetically modified goats through the random gene integration and gene mapping integrate, whose breast can specifically produce salmon calcitonin.In the preparation of random-integrated transgenic goats, we construct a mammary-gland-specific vector named pBLG-sct, which has a promoter from the regulatory region of bovineβ-lactoglobulin. By digestion we get the linearized BLG-sct from pBLG-sct and the linearized neo from pGH4. Then through co-transfecting BLG-sct and neo into goats'fetal fibroblast cells, we obtained 127 cloned cell lines with G418 resistance (14d). Finally, we got 11 positive clones in 15 cell lines in 6-well plates. In the process of nuclear transfer, the fusion rate of sct-neo-8 and sct-neo-9 is 91.04% and 88.43%. Totally, we transplanted 40 receptor ewes (17 for sct-neo-8; 23 for sct-neo-9). Therty days later,11 ewes became pregnant by type-B ultrasonic. At 20th day of pregnancy, one ewe abortioned and the fetus proved to be positive.In order to improve the efficiency of transgene expression, we conduct research on the positioning integration of the sct-breast-specific expression framework. First of all, this study tested the deletion efficiency of cell-permeable Cre in goat fibroblast cells. E. coli BL-21 was induced with IPTG and produced TAT-cre. Then we obtained 1.883mg/ml TAT-cre through SP sepharoseTM. At last, it proved that TAT-cre showed good activity in goat fibroblast cells and its deletion efficiency is up to 43.9% after TAT-cre incubated with sct-neo-integrated clonal cell lines.Finally, we constructed a vector named p2xGsct-zeo with a framework of [loxp-neo-tk-loxp]. Then take the vector into fetal fibroblast cells of the transgenic goats expressing recombinant human lysozyme (rhLYZ) by electroporation transfection. After the trearment of TAT-cre, PCR results showed that specific bands appeared. So the Testing program is feasible, which TAT-cre can make p2xGsct-zeo and the [loxp-neo-tk-loxp] of the transgenic goats expressing recombinant human lysozyme interchangeable.In summary, this study not only won the random integration of breast-specific expression of salmon calcitonin in the framework of the transgenic cells and the fetus, but also confirmed using TAT-Cre can achieve the deletion of marker gen and the integration of exogenous gene mapping in the process of preparation of transgenic goat. These will be used for preparing transgenic goat with highly expressed salmon calcitonin and achieving the integration of gene mapping.