31 CONSTRUCTION OF A TARGETING VECTOR SPECIFIC FOR THE BOVINE BETA-CASEIN GENE

Abstract

Although using livestock as animal bioreactors is a powerful tool to produce valuable therapeutic proteins in the milk, there are still some limitations to the technology such as a low frequency of transgenesis and a low expression of the transgenes by random integration. In this study, we constructed gene-targeting vectors for a mammary gland-specific gene and then obtained two homologous recombinant cell clones after transfection of the vectors into bovine somatic cells. Two targeting vector cassettes, BCKI I and BCKI II, which have homology regions for a bovine beta-casein gene, were constructed. The beta-casein gene is expressed only in the mammary gland and is the most abundant milk protein in the cow. The targeting sequence lengths of the BCKI I and BCKI II vector cassettes were 13.1 kb and 9.1 kb, respectively, and contained different long arm lengths. A neo gene was inserted into the vectors as a selection marker, and a few restriction enzyme sites were made in front of the neo gene. The human thrombopoietin (TPO) gene was inserted into the restriction enzyme sites of the vector cassettes, named BCTPOKI I and BCTPOKI II vectors. The BCTPOKI I and BCTPOKI II vectors were transfected into bovine embryonic fibroblasts (bEF) and ear skin fibroblasts (bESF) using Lipofectamine^™ 2000 reagent (Invitrogen, Seoul, South Korea). In order to determine the highest transfection efficiency, a variety of factors such as DNA concentration, lipid volume, and exposure time to DNA-liposome complexes based on the manufacturers' guideline, was optimized. The 2:1 and 1:2 ratios of DNA (μg) to transfection reagent (μL) were efficient for bEF and bESF, respectively, under overnight exposure to DNA-Lipofectamine^™ 2000 reagent. Seventeen percent (51/304) of bESF clones and 6% (9/149) of bEF clones were normally expanded into passage 8. PCR and Southern blotting indicated that 6.3% (2/32) of the clones carrying with BCTPOKI II vectors was homologously targeted at the beta-casein gene. However, none (0/60) of the clones carrying BCTPOKI I was targeted. Additionally, both of the targeted clones were from bESF. When the targeted cells were transferred into enucleated oocytes and cultured, 83% (43/52) of the cloned embryos were transgenic. Thus, we found that homologous recombinant events using gene-targeting vectors might be dependent on cell types, vector sizes, and transfection procedures. In conclusion, mammary gland-specific gene-targeting vectors coupled with somatic cell nuclear transfer technology will be very useful for developing animal bioreactors that produce therapeutic proteins in milk.