Highly Efficient Generation of GGTA1 Biallelic Knockout Inbred Mini-Pigs with TALENs

Abstract

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

“…Various mutation patterns at the target site were observed, including small deletion (-1 base), large deletions (-226 bases), and small insertions (+1 base), which are characteristic of non-homologous end joining (NHEJ)-induced DNA damage response. The vast majority of mutations were deletions, which were also reported previously in mice and pigs3235. Among all the targeted cell colonies, more than 1/3 were bi-allelic mutations, and this amount was consistent with previous reports in pigs632.…”

“…The vast majority of mutations were deletions, which were also reported previously in mice and pigs3235. Among all the targeted cell colonies, more than 1/3 were bi-allelic mutations, and this amount was consistent with previous reports in pigs632.…”

“…For E-PCR3132, no off-target sites were predicted for the 3 TALEN pairs. Based on the TALENoffer score33, 5-7 potential off-target sites were verified by PCR amplification and sequencing (Supplementary Tables S1–S3 online).…”

RAG1/2 Knockout Pigs with Severe Combined Immunodeficiency

“…Somatic cell nuclear transfer and embryo transfer SCNT was performed as described in previous studies (21,22). Pig ovaries were transported from a local slaughter house to the laboratory in 0.9% saline at 35-39˚C.…”

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…For example, Yang et al (2011) used ZFNs to create pigs with a ppar-γ gene knock-out to study the role of this gene in cardiovascular diseases. A number of works studied the knock-out of the ggta1 gene using ZFNs, TALENs, or CRISPR/Cas; this gene encodes the α-1, 3-galactosyl-transferase generating the Gal epitopes that are responsible for immune rejection of organs in pig-to-human xenotransplantation (Hauschild et al, 2011;Hauschild-Quintern et al, 2013;Li et al, 2013c;Xin et al, 2013;Bao et al, 2014;Yao et al, 2014;Li et al, 2015). Even though those genome modifications are of limited agronomical significance, the knowledge and experience obtained in these studies can help to develop animals with agronomically beneficial traits.…”