124 KNOCKING-IN OF A FOREIGN GENE ON A BOVINE BETA-CASEIN GENE INTO BOVINE PRIMARY FIBROBLASTS USING HOMOLOGOUS RECOMBINATION EVENTS

Knocking-in of a foreign gene on a tissue-specific genomic region has not been reported in livestock. In this study, we constructed two knock-in vector cassettes specific for the bovine beta-casein gene which is expressed only in the mammary gland during lactation. The targeting vector cassettes, pBCKI1 and pBCKI2, have homology regions for the bovine beta-casein gene and contain 13.1 kb and 9.1 kb targeting arms with different long arm lengths, respectively. The targeting vector cassettes have unique restriction enzyme sites for insertion of foreign therapeutic genes in front of the neo gene which was inserted into the vector as a selection marker. The human thrombopoietin (hTPO) gene was inserted into the restriction enzyme sites of both targeting vectors, which were named pBCTPOKI1 and pBCTPOKI2. When the two targeting vectors were transfected into bovine ear skin fibroblasts using Lipofectamine (Invitrogen, Seoul, South Korea), 6.3% of neo resistant clones (2/32) were homologously targeted with the pBCTPOKI2 vector. Cells from the targeted colonies were nuclear-transferred into enucleated bovine oocytes and cultured to the blastocyst stage. A total of 66 blastocysts were generated of which 46 were transferred into recipients. No offspring have been produced at this time. Here we first describe knock-in vectors specific for a bovine beta-casein gene, which will be employed to generate animal bioreactors that produce therapeutic proteins secreted into the milk.