Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.
Stem Cell Reports
; 12(5): 1145-1158, 2019 05 14.
Article
em En
| MEDLINE
| ID: mdl-30956114
ABSTRACT
We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sarcômeros
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Miócitos Cardíacos
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Células-Tronco Pluripotentes Induzidas
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Sistemas CRISPR-Cas
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Edição de Genes
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article