Study On Expression Of Bovine Prolactin Vectors In Mouse Cell Lines

Prolactin (PRL) exists in all known vertebrates. It is an indispensable factor in the mammary development and lactogenesis of mammals. Prolactin can promote the synthesis of various milk proteins. Previous researches show that bovine prolactin increases the expression of exogenous genes in mammalian cells.This thesis clones the promoter of bovine prolactin gene, PRLP, which includes a 2.2kb 5'-flank sequence from the transcription starting site. This sequence contains the segment from -1124bp to -985bp, an important region for effective expression. This thesis utilizes the 9.4kb genomic sequence of bovine prolactin gene (bPRL, GenBank: AF426315) as target gene, and constructs three expression vector with different promoters, CMV promoter (human cytomegalovirus promoter, 800bp), PRLP promoter (bovine prolactin gene promoter, 2.2kb) and P1A3 promoter (goatβ-casein gene promoter, 6.7kb).This thesis transfects AtT20 (mouse pituitary tumor cell strain) and HC11 (mouse mammary epithelial cell strain) cell lines with the three expression vectors constructed. RT-PCR and Realtime RT-PCR methods are adopted to analyze the expression level of the three vectors in both cell lines. pCMV vector is effectively expressed in both cell lines, pPRLP vector has a similar expression level to that of pCMV, pP1A3 is expressed in HC11 but not in AtT20. This thesis produces transgenic mice using the expression vectors constructed. The integrated bovine prolactin gene can be passed to filial generation through germ cells. One bPRL++TF+ double integrated mouse is obtained through mating bPRL+ mouse with human transferrin transgenic mouse.This thesis analyzes the expression level of three vectors in two mammal cells, and therefore compares the regulation of bovine prolactin gene expression by different promoters. The results of this thesis provide information to study the possibility of using bovine prolactin to increase the expression of exogenous genes in transgenic animals.