Highly Efficient Generation Of GGTA1Biallelic Knockout Inbred Mini-pigs With TALENs

Inbred mini-pigs are ideal organ donors for future human xenotransplantationsbecause of their clear genetic background, high homozygosity, and high inbreedingendurance. Hyperacute rejection (HAR) is a major obstacle to pig-to-primatexenotransplantation. Disruption of the-1,3-galactosyltransferase (GGTA1) gene,which is essential for Galactose-1,3-galactose(Gal-1,3Gal or Gal), is the firststep toward overcoming HAR. GGTA1knockout (KO) swine were generated byseveral groups through a combination of traditional DNA homologous recombination(HR) and somatic cell nuclear transfer (SCNT). The gene targeting efficiency oftraditional DNA HR technology is extremely low and most of the KO pigs previouslyreported were difficult to expand because of its low fertility and outbred feature.Transcription activator-like effector nuclease (TALENs), is a new genome-modifyingtechnology. Similar to Zinc-finger nuclease (ZFNs), TALENs can mediate DNAdouble-strand breaks in a specific desired sequence, cause frame-shift mutation, andsilence the expression of target genes at high efficiency. TALENs have advantagesover ZFNs in many aspects, such as in availability, specificity, flexibility and lowertoxicity. TALENs have been successfully applied for efficient gene targeting inseveral animal models. As of this writing, the production of KO pigs with TALENshas been reported only by a few groups. Though the emerging gene modifiedtechnologies have been extremely improved the gene knockout efficiency in the celllevel, the SCNT technology in pig is still characterized by inefficiency and the clonedanimals are at risk of abnomal birth weight, organ deficience, congenital disorders.Alarge-scale cloned pigs could not obtained though SCNT and the group could notexpand due to inbreeding depression.Inbred animals were characterized by cleargenetic background, high homozygosity and high inbreeding endurance.With theinbred animals, once the genetically modified animals were cloned, the populationwith the samegenetic modification could be expanded easily through consanguineousmating without severe depression in convensional animals. In this study, we isolated fetal fibroblast cells from a highly inbred pig line calledBanna mini-pig inbred line (BMI).Seven35-day-old fetuses were obtained and fetalfibroblast cell lines were established. The fetal genders were identified by SRY-PCRmethod. Two pairs of TALENs，TALENs Set1#and TALENs Set2#, targeting exon6of porcine GGTA1were constructed and synthesized. To validate the efficiency ofTALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected intoone-cell stage parthenogenetically activated porcine embryos. The efficiency of indelmutations of TALENs Set1#at the GGTA1-targeting loci was as high as73.1%(19/26) among the parthenogenetic blastocysts. TALENs were co-transfected intoporcine fetal fibroblasts of BMI with pcDNA3.1containing neomycin gene. Thetargeting efficiency of TALENs Set1#reached89.5%(187/209) among the survivedcell clones after8-10d selection. More remarkably27.8%(58/209) of colonies werebiallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nucleardonors for SCNT. Three miniature piglets with biallelic mutations of the GGTA1gene were achieved. Gal epitopes on the surface of cells from all the three biallelicKO piglets were completely absent. The fibroblasts from the GGTA1null pigletswere more resistant to lysis by pooled complement-preserved normal human serumthan those from wild-type pigs.On the basis of generation of GGTA1KO female BMI pigs, to facilitate theexpansion of homozygous GGTA1KO pig population by breeding, we next attemptedto creat male GGTA1KO pigs. A male cell line from BMI was used as donor cells.The targeting efficiency in the survived male cell colonies after transfection andscreening was72.5%.The achieved cloned embryos were transferred into six estroussurrogates and two of them were pregnant. One surrogate aborted one GGTA1KO malepiglet. The other surrogates developed to term and one male piglet with double GGTA1KOhad been achieved. The consanguineous mating among the pigs with the same geneticmodification can be conducted to expand the population.These results indicate that a combination of TALENs technology with SCNT cangenerate biallelic KO pigs directly with high efficiency. The GGTA1null piglets withinbred features created in this study can provide technical basis forxenotransplantation research.