Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase from Porcine Endothelial Cells by a CRISPR System

R. Sakai, Y. Esaki, Hidetoshi Hasuwa, M. Ikawa, P. Lo, R. Matsuura, K. Nakahata, M. Zenitani, M. Asada, A. Maeda, H. Eguchi, H. Okuyama, S. Miyagawa

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Abstract

Background We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Methods Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. Results Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. Conclusions Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.

Original languageEnglish
Pages (from-to)1320-1322
Number of pages3
JournalTransplantation Proceedings
Volume48
Issue number4
DOIs
Publication statusPublished - 2016 May 1
Externally publishedYes

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ASJC Scopus subject areas

  • Surgery
  • Transplantation

Cite this

Sakai, R., Esaki, Y., Hasuwa, H., Ikawa, M., Lo, P., Matsuura, R., Nakahata, K., Zenitani, M., Asada, M., Maeda, A., Eguchi, H., Okuyama, H., & Miyagawa, S. (2016). Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase from Porcine Endothelial Cells by a CRISPR System. Transplantation Proceedings, 48(4), 1320-1322. https://doi.org/10.1016/j.transproceed.2015.10.065