Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection

AbstractPluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.

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Faculty Opinions recommendation of Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1708959.1216057 ◽

2010 ◽

Author(s):

Alexander Ploss

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Human Hepatocyte ◽

Highly Efficient ◽

Efficient Generation ◽

Induced Pluripotent

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Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

Stem Cells Translational Medicine ◽

10.1002/sctm.20-0007 ◽

2020 ◽

Cited By ~ 1

Author(s):

Zhong‐Dong Shi ◽

Jason Tchao ◽

Ling Wu ◽

Aaron J. Carman

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Suicide Gene ◽

Highly Efficient ◽

Induced Pluripotent

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Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

10.1101/2021.02.02.429390 ◽

2021 ◽

Author(s):

Rabea Dettmer ◽

Isabell Niwolik ◽

Ilir Mehmeti ◽

Anne Jörns ◽

Ortwin Naujok

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Beta Cells ◽

Pluripotent Stem Cells ◽

Reporter Genes ◽

Human Pluripotent Stem Cells ◽

Endogenous Gene ◽

Pancreatic Progenitors ◽

Insulin Producing Cells ◽

Endogenous Genes

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

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