High ubiquitous hCD55 and hCD39 co-expression in live transgenic alpha1,3-Gal Knock-out piglet

in xenotransplantation studies (pig to non-human primates) it was demonstrated that alpha-1,3 galactosyltransferase gene deletion (gal-ko) protected transplanted organs from hyperacute rejection. However the vascular acute rejection (avr) has to be solved through genetic engineering of donors inserting different human transgenes involved into prevention of complement activation (hCd55), coagulation and inflammation (hCd39) pathways. we created two different ubiquitous expression vectors for hCd55 (53mdH) and hCd39 (2JC9) exploiting the pCaggs promoter. primary fibroblasts (1x10 6 ) from gal-ko pig were co-transfected with 5 micrograms of 53mdH+2JC9 (1:3) using nucleofector technology. after 24 hours, selection medium (Hygromicine = 175 ug/ml) was added to transfected cells and maintained for 15 days. sixty well growing resistant clones were picked up and analyzed for transgenes expression using western blot (wb) and immunocytochemistry (iCC). about 28% of colonies (n=17) showed high and uniform expression of both transgenes. four colonies were used as cell donors for sCnt and 265 cloned compacted morula and blastocysts d6 were transferred into 3 synchronized recipient sows. all sows became pregnant, 2 aborted at 30 th day, but one went to term (33%) and delivered one alive piglet and mummified fetus. the piglet died for e.coli infection at the age of 1 month. expression hCd39 was detected in every tissue (kidney, pancreas, brain, muscle, heart, liver, spleen, lung, umbilical cord) and primary cells (fibroblasts and porcine aortic endothelial cells) analyzed with wb and iCC whereas hCd55 was not detected only in brain sample. we obtained a gal-ko piglet with ubiquitous high hCd39+hCd55 expression using the pCaggs promoter and he survived for one month without any apparent disorder. more pregnancies are ongoing with the same cell clones. the function of these two transgenes combination will be tested by xenotransplantation to primates as soon as more animals will be available