Construction And Expression Of Marker-free Targeting Vector Of Human Leukemia Inhibitory Factor

With the development of biotechnology, transgenic technology has gradually shifted the use of mammary gland bioreactor for a large number of clinical proteins. Animal mammary gland bioreactor is an organ which can guide the foreign gene expressed. To this end,we can construct specific expression to produce transgenic animals and then get the recombinant protein in animal mammary gland. As it has odds in site-specific integration of foreign genes into the recipient cell genome, overcoming the position effect caused by random integration and multi-copy insert problem, so now the somatic cell gene targeting and nuclear transferring become the main technical options in preparation of animal mammary gland bioreactor. In this study, we insert the hLIF gene into the goatβ-casein gene locus and verify its expression in goat mammary cells layed a foundation for the in-depth study of using mammary gland bioreactor to produce hLIF protein.In this study , first we got a two same sequences of Loxp and insert it to the pBluescript_SK+, then got the pLoxP vector, and nest we clone 5 'homologous arm, 3 'homologous arm, positive selection marker gene neo and negative selection marker gene tk into the pLoxP vector , then we get the pLoxP-NT53vector.At last we clone the hLIF gene into the pLoxP-NT53.In this vector the hLIF gene is at the downstream of the goatβ-casein gene exon VII, neo locus the between the LoxP sequence, tk is at the downstream of 3 'homologous arm.Taking goat mammary tissue as material, primary cells of goat mammary epithelium were cultured by explants adherence method, then obtained purified goat mammary epithelial cells, after the pLoxP-NT53L restriction by SwaⅠ, transfected by Liposome into the goat mammary epithelial cells, resistance to geneticin (G418) and resistance to ganciclovir (GANC) in the cells to get homologous recombination positive monoclonal. Then we verify the hLIF expression by PCR, RT-PCR and Western blotting.The restriction and sequencing analysis result suggested that the targeting vector pLoxP-NT53L is constructed successfully. After detected by PCR and RT-PCR, we got 14 positive clones, and then we induced the clones by hormone, and found that 2 clones can express the hLIF after detected by Western blotting. It is suggest that the hLIF is integrated into theβ-casein locus and the goat mammary cells can express protein specificity. Based on the study, it is very useful for produce marker-free dairy goat of transfer hLIF gene.