Rafael Miyashiro Nunes Dos Santos1, Luiz Augusto Carneiro D'Albuquerque2, Luz M Reyes3, Jose L Estrada3, Zheng-Yu Wang3, Matthew Tector3, A Joseph Tector4. 1. Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana; Digestive Organs Transplant Division, Gastroenterology Department, Sao Paulo University School of Medicine, São Paulo, Brazil. 2. Digestive Organs Transplant Division, Gastroenterology Department, Sao Paulo University School of Medicine, São Paulo, Brazil. 3. Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama. 4. Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama. Electronic address: jtector@uab.edu.
Abstract
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.
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 donorpigs for xenotransplantation. MATERIALS AND METHODS: We used Cas9 to cut pigthrombomodulin (pTHBD) and replace it with a plasmid containing a promoterless antibiotic selection marker and the exon for humanthrombomodulin. 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.
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