The Expression Of Human Lactoferrin In Goat Mammary Epithilial Cells And The Study Of Nuclear Transfer

In this paper,the human lactoferrin gene expressed vector pBLM-C1 which was transfected into goat mammary gland epithelial cell with Liposome。Positive clone cells were selected with G418 and PCR。Then these cells were induced by hormone (prolactin + insulin+ hydrocortisone) to express Human lactoferrin gene。The result of Western blot analysis on cultured cell supernatant showed that transfected cells can express Human lactoferrin gene。The positive cells were used as nucleus donor to create transgenic cloned embryos by nuclear transfer techniques。The foreign gene in transgenic cloned embryos was detected by PCR,lays a solid foundation for constructing transgenic animals and its mammary gland bioreactor。The results of this research were as following:1 The primary cultured goat mammary gland epithelial cell,majority of the cells were short shuttle-like or guboidal which looked like beehive;part of the cells looked like big,round flat cakes;part of the cells were long。The cells could form island-like or dome-like structure in monolayer culture。The transfected experiment was executed when cells were subcultured to the 3th passage。2 The purifyied vectors were mixed in prope ratio with liposome,which was used to transfected cultured of goat mammary gland epithelial cells。High clone rate can be acquired by the liposome transfected method,The island-like structure could formed by positive cells。3 The transfected goat mammary gland epithelial cells were selected by G418 400μg/mL for two weeks,then were cultured in G418 200μg/mL。the transfected cells DNA were abstracted and be identifyied by PCR,the results revealed that this vector was transfected into cells successfully。4 The transfected cells were induced by hormone (prolactin + insulin+ hydrocortisone) to express Human lactoferrin gene。The supernatant was collected 1 time every 6hs,then was centrifuged for 10～15min (1000r/min),which was stored in–20℃。The result of Western blot analysis on cultured cells supernatant showed that transfected cells can express Human lactoferrin protein,the molecular weight is 42ku。5 The transgenic cloned embryos were created effectively by means of electro-fusion,...