Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73

Abstract

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Developmental and Degenerative Characterization of Porcine Parthenogenetic Fetuses during Early Pregnancy

“…Parthenogenesis is distinct from pseudo-pregnancy, and we hypothesized that PA embryos have contributed to the pregnancy-live birth gap with ART. In our previous reports, the early pregnancy of transgenic cloned pigs was 32% in alpha 1,3-galactosyltrasnferase knock-out with membrane cofactor protein knock-in and 44% in human CD73 transgenic, but the delivery rate was 16% and 17%, respectively [17,18].…”

Bioinformatic analysis as a first step to predict the compatibility of hematopoiesis and immune system genes between humans and pigs

“…Therefore, these dissimilar genes should be paid attention to avoid coagulation disorders in human and pig xenogeneic combination settings. There are several important studies reporting the function similarities or dissimilarities of related proteins between humans and pigs, which are summarized with their sequence similarities and domain composition consistency identified in the present study [20][21][22][61][62][63][68][69][70][71][72][73][74][75][76][77][78][79][80] (Table 1).…”

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum

“…The ICAM2 promoter has been frequently used for specific expression of anti-coagulation and anti-inflammatory proteins, including tissue factor pathway inhibitor (TFPI), thrombomodulin (THBD), C1 inhibitor, and A20 [ 3 , 19 , 20 ]. In previous studies, we reported the production of GTKO pigs carrying human CD46 (GTKO/CD46) or THBD (GTKO/CD46/THBD) driven by the CMV promoter [ 21 , 22 ], CAG promoters for ubiquitous expression, and the pig ICAM2 promoter for endothelium-specific expression of THBD and CD73 [ 23 ]. We demonstrated that the CMV promoter was moderately active for CD46 expression in GTKO/CD46 pigs, and the CAG promoter was rather strong for CD46 expression in GTKO/CD46/THBD pigs.…”