Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.
Circ Res
; 117(2): 121-8, 2015 Jul 03.
Article
em En
| MEDLINE
| ID: mdl-25940550
ABSTRACT
RATIONALE The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. OBJECTIVE:
To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. METHODS ANDRESULTS:
To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression.CONCLUSIONS:
CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Proteínas Nucleares
/
Transativadores
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Deleção de Genes
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Lentivirus
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Técnicas de Inativação de Genes
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Sangue Fetal
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Sistemas CRISPR-Cas
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Células Progenitoras Endoteliais
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Vetores Genéticos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Female
/
Humans
Idioma:
En
Revista:
Circ Res
Ano de publicação:
2015
Tipo de documento:
Article