Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (2): 281-285.doi: 10.3969/j.issn.2095-4344.2958

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Development and evaluation of alpha-galactosyl antigen-deficient rabbit model

Mu Yufeng1, 2, Wei Lina1, Wu Yong1, Shao Anliang1, Chen Liang1, Qu Shuxin2, Xu Liming1, 2     

  1. 1National Institutes for Food and Drug Control, Beijing 102629, China; 2School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China
  • Received:2020-01-14 Revised:2020-01-19 Accepted:2020-03-09 Online:2021-01-18 Published:2020-11-21
  • Contact: u Liming, MD, Researcher, National Institutes for Food and Drug Control, Beijing 102629, China; School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China Qu Shuxin, PhD, Professor, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China
  • About author:Mu Yufeng, Master candidate, National Institutes for Food and Drug Control, Beijing 102629, China; School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China Wei Lina, MD, Assistant research, National Institutes for Food and Drug Control, Beijing 102629, China
  • Supported by:
     the National Key Research and Development Project of China, No. 2016YFC1103203

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Abstract:

BACKGROUND: α-Galactosyl (α-Gal) is the main target antigen in hyperacute rejection resulting from animal tissues or xenotransplantation. 
OBJECTIVE: To develop a Gal antigen-deficient rabbit model in order to objectively evaluate the immunogenicity risk of animal-derived biomaterials and the local response to implantation in the host. 
METHODS: New Zealand white rabbits, SPF grade, 6-8 months old, were selected as model animals to prepare Gal antigen-deficient rabbits. Using CRISPR/Cas9-mediated gene editing technology, two complementary sgRNAs were designed and constructed for the 8th exon of GGTA1 gene to regulate Gal antigen expression in rabbits. After transcription, GGTA1 sgRNA mRNAs and Cas9 mRNA were co-microinjected into in vitro cultured rabbit fertilized eggs, which were then implanted into surrogate mother rabbits after a brief in vitro culture, and the neonatal rabbits were obtained by natural pregnancy. The success of gene editing was verified by gel electrophoresis and gene sequencing. The expression of Gal antigen was detected with reference to the method given by the industry standard (YY/T 1561-2017). The study protocol was approved by the Animal Ethics Committee of the Laboratory Animal Resources Laboratory of the National Institutes for Food and Drug Control (approval No. 2017(B)007).
RESULTS AND CONCLUSION: The embryos were transferred to four surrogate rabbits after gene editing. FIfteen gene-edited pups were obtained after natural pregnancy. The appearance and feeding behavior of the pups were not abnormal. Gel electrophoresis and gene sequencing results showed that 14 out of 15 rabbits were successfully edited, but the edited bases were not the same. The Gal antigen of the main organs was detected randomly, and its expression was reduced by more than 99.96%. Therefore, Gal-antigen-deficient rabbits are expected to be used for immunogenic risk assessment of animal-derived medical devices and in situ implantation experiments of heterogeneous bone and cornea, in order to be able to more objectively and scientifically evaluate the safety and effectiveness of animal-derived medical devices.

Key words: Gal antigen, GGTA1 gene, CRISPR/Cas9, gene editing, immune rejection, model animals, orthotopic implantation, Gal antigen-deficient rabbit

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