CRISPR/Cas and recombinase-based human-to-pig orthotopic gene exchange for xenotransplantation
| Autor: | Luz M. Reyes, Rafael Miyashiro Nunes dos Santos, Luiz Augusto Carneiro D'Albuquerque, Matthew Tector, A. Joseph Tector, Zheng-Yu Wang, Jose Estrada |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Swine Thrombomodulin Xenotransplantation medicine.medical_treatment Transgene Primary Cell Culture Transplantation Heterologous 030230 surgery Biology Transfection Animals Genetically Modified Recombinases Viral Proteins 03 medical and health sciences 0302 clinical medicine Plasmid Genome editing Recombinase medicine Animals CRISPR Bacteriophages DNA RECOMBINANTE Gene Cells Cultured Gene Editing Regulation of gene expression Endothelial Cells Cell biology 030104 developmental biology Tissue and Organ Harvesting Surgery CRISPR-Cas Systems |
| Zdroj: | Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 0022-4804 |
| DOI: | 10.1016/j.jss.2018.03.051 |
| Popis: | Background Tools for genome editing in pigs are improving rapidly so that making precise cuts in DNA for the purposes of deleting genes is straightforward. Development of means to replace pig genes with human genes with precision is very desirable for the future development of donor pigs for xenotransplantation. Materials and methods We used Cas9 to cut pig thrombomodulin (pTHBD) and replace it with a plasmid containing a promoterless antibiotic selection marker and the exon for human thrombomodulin. PhiC31 recombinase was used to remove the antibiotic selection marker to create porcine aortic endothelial cells expressing human instead of pTHBD, driven by the endogenous pig promoter. Results The promoterless selection cassette permitted efficient enrichment of cells containing correctly inserted transgene. Recombinase treatment of selected cells excised the resistance marker permitting expression of the human transgene by the endogenous pTHBD promoter. Gene regulation was maintained after gene replacement because pig endogenous promoter was kept intact in the correct position. Conclusions Cas9 and recombinase technology make orthotopic human for pig gene exchange feasible and pave the way for creation of pigs with human genes that can be expressed in the appropriate tissues preserving gene regulation. |
| Databáze: | OpenAIRE |
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