A Preliminary Study On The Construction Of TPO Targeting Vector Expresed In Mammal Gland

The low expression level of purpose gene is the main problem in the current study of mammary gland bioreactor, due to the random insertion and position effects of integrated genes. The development of gene targeting and animal cloning thechnology makes the site-specific integration of foreign gene become possible. Thus, the present study was undertaken to explore the possibility of constructing thrombopoietin(TPO) gene targeting vector specially expressed in mammary gland.The gene targeting vector was planning to be constructed by using Thrombopoietin Gene (TPO) gene as purpose gene, as 1-casein gene as targeting gene, the 5' sequence of β-casein gene as promotor. Firstly, 4.1kb 5' sequence of sheep and buffalo β-casein gene was amplified by the long distance-PCR technique. The segment contains the sequence from the far upstream sequence to the part of exon2. Then, 5.5kb thrombiotin gene was amplified with the same technique from the genome of a baby's blood, which included the begining part of intronl to the teminator. In addition, 6.0kb and 1.8kb homlogous arms were also amplified from a cow with high yield. The 6.0kb homologous arm contains the promotor, extron 1, extron2, extron3 and intron 1, intron2 and part of the intron3 fragment, while the 1.8kb homologous right arms contains exon13, exon14 and part of intron 13, the whole intron14 and part intron 14 of asl-casein gene of bovine. All these fragments were cloned into pMD-18T vector for the enzyme and sequencing analysis. Enzyme cutting analysis indicated the enzyme sites suport to establish a targeting vector. Homologous sequence aligmemt shown 99% homologous of all fragments to the template animal or human DNA sequence published in Genebank, indicating that all the fragments are suitable for constructing the targeting vector.