Abstract：In this study we construct a maker-free targeting vector of human leukemia inhibitory factor and verify its expression in goat mammary cells. First we use the recombination cassette LoxP sequence, goat β-casein 5′and 3′ homologous arms, positive and negative selection marker cassette to get targeting vector of human leukemia inhibitory factor. Transfection of the vector into goat mammary cells with LipofectamineTM 2000 conferred 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 resulst suggested that the targeting vector pLoxP-NT53L is constructed successfully, and after detected by PCR, RT-PCR and Western blotting, we find the target band, which demonstrate the targeting vector pLoxP-NT53L is integrated into the β-casein locus and the goat mammary cells can express protein specificity. Based on the study, it is very useful for produce marker-free darily goat of transfer hLIF gene.
