Production of ULBP1-KO pigs with human CD55 expression using CRISPR technology

Journal of Applied Animal Research

Jacek Jura¹  Barbara Gawrońska²  Daniel Lipiński²  Natalia Mazurkiewicz²  Magdalena Hryhorowicz²  Joanna Zeyland²  Agnieszka Nowak-Terpiłowska²  Wojciech Juzwa³  Ryszard Słomski⁴ 

[1] Department of Animal Reproduction, National Research Institute of Animal Production;Department of Biochemistry and Biotechnology, Poznan University of Life Sciences;Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences;Institute of Human Genetics, Polish Academy of Sciences;

关键词: crispr-cas9;    ul-16 binding protein;    pigs;    xenotransplantation;    cd55 (daf);   

DOI  :  10.1080/09712119.2020.1735396

来源: DOAJ

【 摘 要 】

The shortage of organs for transplantation is a well-known issue of modern medicine. The domestic pig has proved to be most suitable for xenotransplantation purposes. It is necessary to modify the pig genome to eliminate the immunological differences resulting from its phylogenetic remoteness from humans. We present a generation of pigs with human CD55 and ULBP1 gene knockout. Both modifications were introduced using the CRISPR/Cas9 system. A mixture of Cas9-D10A mRNA, a pair of sgRNAs and the pCD55 donor vector were introduced into the pronucleus of the fertilized porcine oocyte. After microinjection the three surrogates delivered a total of 13 piglets. The analysis showed four piglets with indels in the targeting site of exon 2 of the pULBP1 gene. All four pigs were altered in a biallelic manner, showing different sequences in each mutant allele. One piglet also showed one allele interrupted with a CD55-expressing cassette. The analyses confirmed the integration and the expression of the CD55 transgene in the targeted site. The human serum cytotoxicity test results showed that the highest viability of modified cells was 84.42% compared to the control. The cytometric analysis suggests that the CD55-expressing cassette was integrated with the genome in the transcription active site.

【 授权许可】

Unknown