The Preliminary Study Of Human Serum Albumin Knockin Cloned Pigs By Gene Targeting

Human serum albumin (HSA) is a globular, single band protein of 585 amino acids, which is abundant in blood serum and plays an important role in clinic. By the study of HSA gene knockin porcine serum albumin gene locus and nuclear transfer of targeted cells, we hope to get HSA from porcine blood. We choose pig as our object because of its characteristic such as large capacity of blood, easy to breed and so on. Moreover we can not only avoid the contamination of blood from different virus, but also decrease the cost by obtain the transgenic pig by knockin which has the stable expression ability of HSA in offspring.The porcine serum albumin genomic sequence of 35kb had been cloned and the founction of 5'regulation sequence was studied firstly. It's found that the role of the upstream regulation regions which were at -1.3kb to -1 and at -7.2 kb to -1 was possibly the same.To optimize the construct of gene targeted vector, the plasmid pEXp11 was constructed with the positive select gene neo inserted into the intron of 3 'regulation sequence. The expression of EGFP was regulated by the 5'and 3 'regulation sequence of porcine serum albumin gene. The green fluorescence was observed and the RT-PCR of genomic DNA was done. It was found that the neo could be removed in the course of translation.Two targeted vectors pPHA1 and pPHA2 which have positive and negtive selective genes were constructed using partial porcine serum albumin(PSA) genomic sequence as homologous arms, HSA minigene and HSA cDNA were the interesting genes. Linearized targeting construct DNA (pPHAl) were introduced into porcine fetal fibroblast cells by electroporation. The genomic DNA of anti-drug clones was analyzed by PCR and Southern blotting. Three clones of targeted fetal fibroblast...