CRISPR/Cas and recombinase-based human-to-pig orthotopic gene exchange for xenotransplantation.
J Surg Res
; 229: 28-40, 2018 09.
Article
em En
| MEDLINE
| ID: mdl-29937002
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 ANDMETHODS:
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.Palavras-chave
Texto completo:
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Bases de dados:
MEDLINE
Assunto principal:
Suínos
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Transplante Heterólogo
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Trombomodulina
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Coleta de Tecidos e Órgãos
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Edição de Genes
Limite:
Animals
Idioma:
En
Revista:
J Surg Res
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Brasil