Transgenic rabbits as a model organism for production of human factor VIII

Full text: Here we report the generation of transgenic rabbits as a model species for testing the expression of human clotting factor VIII (hFVIII) in the mammary gland. Human factor VIII is a very complex and large protein whose expression is difficult as the hFVIII requires extensive posttranslational modifications to be biologically active. First transgenic pigs, where the expression of the hFVIII cDNA was targeted to the mammary gland, produced 0.62U/ml of rhFVIII in their milk. Expression of recombinant hFVIII in the transgenic sheep was also achieved albeit at low levels [2]. In the present study we used for microinjection a fusion gene construct consisting of 2.5kb murine whey acidic protein (mWAP) promoter, 7.2 kb cDNA of the hFVIII, and 4.6 kb of 3' flanking sequences of mWAP gene. Totally 130 microinjected zygotes were transferred into recipients and 30 offsprings were delivered. The pups were screened for the transgene by PCR of DNA isolated from ear, and results were confirmed by Southern blot analysis. The transgene was identified in one female founder animal, and it was transmitted to the offspring in a Mendelian fashion. These data demonstrate a stable integration of the gene construct into the germline of transgenic rabbits. Mammary gland biopsies were taken from lactating founder and her female offspring rabbits on the day 20 of lactation. The presence of hFVIII mRNA is currently under detection by RTPCR. Further studies to analyze level of FVIII expression in further lactations are being carried out. (author)