Production of α1,3-Galactosyltransferase-Knockout Cloned Pigs Expressing Human α1,2-Fucosylosyltransferase1

Abstract

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.

Expression of X‐linked genes in deceased neonates and surviving cloned female piglets

“…Pigs have been regarded as promising donors for xenotransplantation and attempts have been made to genetically modify the porcine genome through somatic cell NT in order to make the porcine organs immunologically more compatible to human Kolber-Simonds et al 2004;Lai et al 2002;Ramsoondar et al 2003). The production of pigs through NT has been particularly difficult, probably due to the inefficiencies of in vitro oocyte maturation and embryo culture, and the necessity for at least four good-quality embryos to establish a pregnancy.…”

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

“…Recent studies that disrupted genes in somatic cells for nuclear transfer have used positive selection only using Neo r gene (McCreath et al, 2000;Denning et al, 2001;Lai et al, 2002;Ramsoondar et al, 2003). Large variations in the number of targeted colonies versus the number of random-integrated colonies (1/20 to 1/500) for gene targeting in somatic cells have been reported (Denning et al, 2001;Dai et al, 2002;Ramsoondar et al, 2003).…”

“…Nuclear transfer of somatic cells in pigs is the most promising procedure to achieve knockout of α-1,3-galactosyl transferase (13-GT) gene for pigto-human xenotransplatation (Betthanser et al, 2000;Onishi, et al, 2000;Polejaeva, et al, 2000;Harrison et al, 2002). Production of viable pig and sheep with targeted insertion at several gene loci following nuclear transfer with gene-targeted cells has been reported (McCreath et al, 2000;Denning et al, 2001;Dai et al, 2002;Lai et al, 2002;Phelps et al, 2003;Ramsoondar et al, 2003).…”

Development of pig cloning studies: past, present and future

“…Somatic cell nuclear transfer was used to produce a total of 90 live piglets from in vitro cultured: foetal fibroblast cells (Onishi et al, 2000;Park et al, 2001c;Boquest et al, 2002;Dai et al, 2002;De Sousa et al, 2002;Lai et al, 2002a,b;Walker et al, 2002;Hyun et al, 2003;Ramsoondar et al, 2003;Yin et al, 2003), skin fibroblasts (Bondioli et al, 2001), ear-derived fibroblasts (Park et al, 2002;Lee et al, 2003b), mural granulosa cells (Polejaeva et al, 2000), cells of an indefinite phenotype isolated from foetuses and their genital ridges (Betthauser et al, 2000), and heart cells (probably cardiomyocytes; Yin et al, 2002). In the experiments aimed at determining the in vitro developmental abilities of reconstructed porcine embryos to compact morula and blastocyst stages, foetal fibroblasts (Tao et al, 1999a,b;Koo et al, 2000;Uhm et al, 2000;Verma et al, 2000;Kuhholzer et al, 2000Kuhholzer et al, , 2001Lai et al, 2001;Park et al, 2001b) or cumulus/ mural granulosa cells (Cheong et al, 2000;Uhm et al, 2000;Park et al, 2001a;Martinez Diaz et al, 2002;Nagashima et al, 2002;Lee et al, 2003b;Samiec et al, 2003a,b) as well as ear skin fibroblasts of adult animals (Miyoshi et al, 2001;Roh and Hwang, 2002) were most often used as a source of donor nuclei.…”

“…In the first system cell nuclei at G1 or G0 stage are introduced into enucleated, nonactivated Met II oocytes and simultaneously obtained clonal nuclear-cytoplasmic hybrids are activated (Uhm et al, 2000;Verma et al, 2000;Kuhholzer et al, 2001;Park et al, 2001b;Dai et al, 2002;Lai et al, 2002a,b;Hyun et al, 2003). In the second system cell nuclei at G1, G1/G0 or G2/M stages are introduced into Met II ooplasts, which are activated 30 minutes to several hours (3-4 hours at the most) after nuclear transfer (Betthauser et al, 2000;Kuhholzer et al, 2000;Onishi et al, 2000;Bondioli et al, 2001;Lai et al, 2001;Park et al, 2001a;Boquest et al, 2002;De Sousa et al, 2002;Hyun et al, 2003;Lee et al, 2003b;Ramsoondar et al, 2003;Samiec et al, 2003a,b). Microsurgical transfer of somatic nuclei can be an alternative method for clonal nuclear-cytoplasmic hybrid reconstruction (Tao et al, 1999;Kuhholzer et al, 2000;Onishi et al, 2000;Uhm et al, 2000;Park et al, 2001a;Nagashima et al, 2002;Samiec et al, 2003a,b) prior to cell fusion induced in the electric field.…”