Generation of α1,3-galactosyltransferase and cytidine monophospho-<i>N</i>-acetylneuraminic acid hydroxylase gene double-knockout pigs

Abstract

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Oocyte‐activating capacity of fresh and frozen–thawed spermatids in the common marmoset (Callithrix jacchus)

“…Mouse oocytes are known to respond to strontium chloride very efficiently and this is one of the reasons for the efficacy of ROSI in this species (Ogonuki et al, 2011). For farm animals such as pigs and bovines, electric pulses or calcium ionophores (ionomycin or A23187) are often used for activating oocytes reconstructed by somatic cell nuclear transfer (De Bem et al, 2017;Miyagawa et al, 2015). Such stimuli also enhanced the development of ICSI-derived embryos in these species (Probst & Rath, 2003;Suttner et al, 2000).…”

The desirable donor pig to eliminate all xenoreactive antigens

“…This immediately preceded dramatic improvements in targeted gene inactivation technologies such as zinc‐finger nucleases, transcription activator‐like effector nucleases, and the CRISPR/Cas9 system. These tools enabled CMAH‐only knockout (KO) and GGTA1/CMAH double KO animals to be created within three years of isolating the gene . Peripheral blood mononuclear cells (PBMC) isolated from pigs deficient in both GGTA1 and CMAH activity exhibited reduced human antibody binding compared to GGTA1 KO pig PBMC …”

CRISPR editing in biological and biomedical investigation

“…Transplantation of hearts from GGTA1 knock‐out (KO) pigs into baboons extended the survival process of pig hearts in baboons to 179 days, which was associated with the prevention of hyperacute rejection (Kuwaki et al, ). Then, in order to repress severe acute humoral xenograft rejection, which is critical to prolong the survival of GGTA1 KO pig's kidney in baboons (Chen et al, ), cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH)/GGTA1 double KO pigs were created using ZFNs, TALENs, or CRISPR/Cas9 (Fischer et al, ; Miyagawa et al, ), and the xenoantigenicity was further reduced compared with that of the only GGTA1 KO pigs (Lutz et al, ). On the other hand, to eliminate the risk of in vitro transmission of PERVs to human cells, sixty‐two copies of porcine endogenous retroviruses (PERVs) were simultaneously disrupted by the CRISPR/Cas9 system in PK15 cells, which is admired as another robust application of CRISPR technology in modifying the pig genome for xenotransplantation (Yang et al, ).…”