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

Research output: Contribution to journalArticle

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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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Cytidine
Heterophile Antigens
Swine
Endothelial Cells
Organism Cloning
Plasmids
CMPacetylneuraminate monooxygenase
Genome
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Surgery
  • Transplantation

Cite this

Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase from Porcine Endothelial Cells by a CRISPR System. / Sakai, R.; Esaki, Y.; Hasuwa, Hidetoshi; Ikawa, M.; Lo, P.; Matsuura, R.; Nakahata, K.; Zenitani, M.; Asada, M.; Maeda, A.; Eguchi, H.; Okuyama, H.; Miyagawa, S.

In: Transplantation Proceedings, Vol. 48, No. 4, 01.05.2016, p. 1320-1322.

Research output: Contribution to journalArticle

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, vol. 48, no. 4, pp. 1320-1322. https://doi.org/10.1016/j.transproceed.2015.10.065
Sakai, R. ; Esaki, Y. ; Hasuwa, Hidetoshi ; Ikawa, M. ; Lo, P. ; Matsuura, R. ; Nakahata, K. ; Zenitani, M. ; Asada, M. ; Maeda, A. ; Eguchi, H. ; Okuyama, H. ; Miyagawa, S. / Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase from Porcine Endothelial Cells by a CRISPR System. In: Transplantation Proceedings. 2016 ; Vol. 48, No. 4. pp. 1320-1322.
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T1 - Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase from Porcine Endothelial Cells by a CRISPR System

AU - Sakai, R.

AU - Esaki, Y.

AU - Hasuwa, Hidetoshi

AU - Ikawa, M.

AU - Lo, P.

AU - Matsuura, R.

AU - Nakahata, K.

AU - Zenitani, M.

AU - Asada, M.

AU - Maeda, A.

AU - Eguchi, H.

AU - Okuyama, H.

AU - Miyagawa, S.

PY - 2016/5/1

Y1 - 2016/5/1

N2 - 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.

AB - 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.

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