Possible detrimental effects of beta‐2‐microglobulin knockout in pigs

Sake, Hendrik Johannes; Frenzel, Antje; Lucas-Hahn, Andrea GND; Nowak-Imiałek, Monika Anna GND; Hassel, Petra GND; Hadeler, Klaus Gerd GND; Hermann, Doris GND; Becker, Roswitha; Eylers, Heinke; Hein, Rabea; Baars,Wiebke; Brinkmann, Antje; Schwinzer, Reinhard; Niemann, Heiner GND; Petersen, Björn GND

Background Despite major improvements in pig‐to‐primate xenotransplantation, long‐term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. Methods In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M‐KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. Results Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. Conclusion Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta‐2‐microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

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Sake, Hendrik / Frenzel, Antje / Lucas-Hahn, Andrea / et al: Possible detrimental effects of beta‐2‐microglobulin knockout in pigs. 2019.

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