Abstract
Donor major histocompatibility complex class I (MHC I) molecules are the main targets of the host immune response after organ allotransplantation. Whether and how MHC I-deficiency of pig donor tissues affects rejection after xenotransplantation has not been assessed. Beta2-microglobulin (B2M) is indispensable for the assembly of MHC I receptors and therefore provides an effective target to disrupt cell surface MHC I expression. Here, we report the one-step generation of mutant pigs with targeted disruptions in B2m by injection of porcine zygotes with B2m exon 2-specific TALENs. After germline transmission of mutant B2m alleles, we obtained F1 pigs with biallelic B2m frameshift mutations. F1 pigs lacked detectable B2M expression in tissues derived from the three germ layers, and their lymphocytes were devoid of MHC I surface receptors. Skin grafts from B2M deficient pigs exhibited remarkably prolonged survival on xenogeneic wounds compared to tissues of non-mutant littermates. Mutant founder pigs with bi-allelic disruption in B2m and B2M deficient F1 offspring did not display visible abnormalities, suggesting that pigs are tolerant to B2M deficiency. In summary, we show the efficient generation of pigs with germline mutations in B2m, and demonstrate a beneficial effect of donor MHC I-deficiency on xenotransplantation.
Highlights
Pigs are considered promising candidate donors for xenotransplantation because they share many anatomical and physiological features with humans, and have a large litter size and a relatively short gestational period
To disrupt B2M expression in pig, we designed a pair of Transcription activator-like effector nucleases (TALENs) molecules that target exon[2] of pig B2m (Fig. 1a). These B2m-TALENs did not have any predicted off-target sites (OTs) in the pig genome according to UCSC In-Silico PCR software
To assess B2m-TALEN-affected early stage development in pig embryos, we injected mRNAs encoding the TALEN pair (10 ng/mL each) into the cytoplasm of parthenogenetically activated (PA) pig embryos at the one-cell stage, followed by culture of embryos in vitro to the blastocyst stage
Summary
Design and validation of TALENs targeting B2m. To disrupt B2M expression in pig, we designed a pair of TALEN molecules that target exon[2] of pig B2m (Fig. 1a). Sequencing of PCR products from the 5 embryos with T7EN1 cleavage bands identified indels and overlapping peaks in sequencing chromatographs (Fig. 1d,e; Table 2), indicating that the B2m-TALENs cleaved the target site. These data demonstrate that TALENs targeting pig B2m had no adverse effects on pig embryo development in vitro, and exhibited target-specific nuclease activity. The two founder pigs (#T1 and #T5, Table 4), in which frameshift, but no other types of mutations were detected and affected both alleles, indicated by the absence of a wild-type allele, did not exhibit any obvious phenotypic abnormalities and grew normally into adults, suggesting that the lack of a B2M molecule had no apparent adverse effects on the development or health of these animals. No additional bands were detectable in PCR reactions amplifying across the B2mt arget area, and T7EN1 cleavage bands were detected
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