Production of Transgenic Goats, Carrying Human Coagulation Factor IX cDNA, by Nuclear Transfer of Transfected Fetal Fibroblasts

There are a growing number of recombinant proteins that have been expressed in milk and one can consider putting any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Nuclear transfer (NT) using donor cells are an efficient means for generation of transgenic animals. In this study in order to produce a transgenic goat with capability of secretion of recombinant human coagulation factor IX (rhcfIX) in the milk, a gene construct (pBC1-hfIX) was made by insertion of (rhcfIX) cDNA after goat beta-casein promoter into the expression cassette. Then, cell lines of goat fetal and adult fibroblasts were transfected by pBC1-hfIX. Fetal fibroblast cells isolated from day 35 to day 40 fetuses. Next, positive clones (the fibroblast cells that contain the gene construct) were selected and confirmed by PCR. The transfected fetal fibroblast cells were allowed to reach confluency and further cultured in 15% FBS in DMEM for an additional 72 h (full confluency) to synchronize G0/G1 cell cycle stage cells. Immature oocytes were obtained from slaughterhouse and matured before enucleation and NT. The reconstructed oocytes electrofused with donor somatic cells, and activated with ionomycin and 6DMAP. Reconstructed embryos were transferred into synchronized recipients. A total of 64 embryos derived from transgenic fetal fibroblast cells were transferred into 12 recipients. Two recipients (16.6%) were confirmed pregnant at Day 42 by ultrasound. Of these, two kids derived from the fetal fibroblast cell line. Presence of the coagulation factor IX gene was confirmed by polymerase chain reaction, sequencing, and fluorescent in situ hybridization analyses. These results demonstrate that goat fetal fibroblasts with human coagulation IX factor gene can direct full-term development following NT. This is the first live born of transgenic farm animal in Iran.