Large Scale Production of Recombinant Human Antithrombin III (rhATIII) in Transgenic Cloned Goats

Abstract:

We report the production of recombinant human antithrombin Ⅲ (rhATⅢ) in transgenic cloned goats. Human ATⅢ mRNA was isolated from human liver tissues, then transcripted into cDNA. The human ATⅢ cDNA (without the first 96 base pair sequence)and Enterokinase recognised peptide-DNA sequence were ligated to a goat beta-casein promoter and poly(A) singling sequences, neomycin selection gene was linked at the end of the poly(A) singling region. The mammary expression construct was transfected into in vitro cultured goat foetal fibroblast cells and the cells were selected with 500ug/ml G418. Five transgenic cloned goats were derived by nuclear transplantation and their transgenic status were confirmed by Southern and/or PCR analysis. The first cloned goat died at 78 day old with abnormalities in respiratory system and kidney. Two of the male cloned goats were bred to produce female offspring. Milk analysis from the transgenic lines revealed that the size of the rhATⅢ in milk was about 60 kD. One line could produce rhATⅢ at 0.4 mg/L in their milk; another line could produce rhATⅢ at a large scale of 3 g/L in their milk. rhATⅢ was also a glycoprotein, which was similar to human serum ATⅢ.