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Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes... Full description

Journal Title: Nature biotechnology 2009, Vol.27 (9), p.851-857
Main Author: Amora, Ranier
Other Authors: Miller, Jeffrey C , Mitalipova, Maisam , Katibah, George E , Rebar, Edward J , Urnov, Fyodor D , Jaenisch, Rudolf , Gregory, Philip D , Boydston, Elizabeth A , Beard, Caroline , Hockemeyer, Dirk , Zeitler, Bryan , Meng, Xiangdong , Soldner, Frank , Gao, Qing , Zhang, Lei , DeKelver, Russell C
Format: Electronic Article Electronic Article
Language: English
Subjects:
Publisher: New York, NY: Nature Publishing Group
ID: ISSN: 1087-0156
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title: Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases
format: Article
creator:
  • Amora, Ranier
  • Miller, Jeffrey C
  • Mitalipova, Maisam
  • Katibah, George E
  • Rebar, Edward J
  • Urnov, Fyodor D
  • Jaenisch, Rudolf
  • Gregory, Philip D
  • Boydston, Elizabeth A
  • Beard, Caroline
  • Hockemeyer, Dirk
  • Zeitler, Bryan
  • Meng, Xiangdong
  • Soldner, Frank
  • Gao, Qing
  • Zhang, Lei
  • DeKelver, Russell C
subjects:
  • Biological and medical sciences
  • Biotechnology
  • Cell Line
  • Deoxyribonucleases - genetics
  • Deoxyribonucleases - metabolism
  • Embryonic stem cells
  • Embryonic Stem Cells - physiology
  • Fundamental and applied biological sciences. Psychology
  • Gene Expression
  • Gene Silencing
  • Gene targeting
  • Gene Targeting - methods
  • Genetic aspects
  • Genetic engineering
  • Green Fluorescent Proteins - genetics
  • Green Fluorescent Proteins - metabolism
  • Homeodomain Proteins - genetics
  • Homeodomain Proteins - metabolism
  • Humans
  • Immunohistochemistry
  • Nucleases
  • Octamer Transcription Factor-3 - genetics
  • Octamer Transcription Factor-3 - metabolism
  • Physiological aspects
  • Pluripotent Stem Cells - physiology
  • Recombinant Fusion Proteins - genetics
  • Recombinant Fusion Proteins - metabolism
  • Stem cells
  • Transcription Factors - genetics
  • Transcription Factors - metabolism
  • Usage
  • Zinc
  • Zinc finger proteins
  • Zinc Fingers - physiology
ispartof: Nature biotechnology, 2009, Vol.27 (9), p.851-857
description: Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
language: eng
source:
identifier: ISSN: 1087-0156
fulltext: no_fulltext
issn:
  • 1087-0156
  • 1546-1696
url: Link


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titleEfficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases
creatorAmora, Ranier ; Miller, Jeffrey C ; Mitalipova, Maisam ; Katibah, George E ; Rebar, Edward J ; Urnov, Fyodor D ; Jaenisch, Rudolf ; Gregory, Philip D ; Boydston, Elizabeth A ; Beard, Caroline ; Hockemeyer, Dirk ; Zeitler, Bryan ; Meng, Xiangdong ; Soldner, Frank ; Gao, Qing ; Zhang, Lei ; DeKelver, Russell C
creatorcontribAmora, Ranier ; Miller, Jeffrey C ; Mitalipova, Maisam ; Katibah, George E ; Rebar, Edward J ; Urnov, Fyodor D ; Jaenisch, Rudolf ; Gregory, Philip D ; Boydston, Elizabeth A ; Beard, Caroline ; Hockemeyer, Dirk ; Zeitler, Bryan ; Meng, Xiangdong ; Soldner, Frank ; Gao, Qing ; Zhang, Lei ; DeKelver, Russell C
descriptionRealizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
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subjectBiological and medical sciences ; Biotechnology ; Cell Line ; Deoxyribonucleases - genetics ; Deoxyribonucleases - metabolism ; Embryonic stem cells ; Embryonic Stem Cells - physiology ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Gene Silencing ; Gene targeting ; Gene Targeting - methods ; Genetic aspects ; Genetic engineering ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Homeodomain Proteins - genetics ; Homeodomain Proteins - metabolism ; Humans ; Immunohistochemistry ; Nucleases ; Octamer Transcription Factor-3 - genetics ; Octamer Transcription Factor-3 - metabolism ; Physiological aspects ; Pluripotent Stem Cells - physiology ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Stem cells ; Transcription Factors - genetics ; Transcription Factors - metabolism ; Usage ; Zinc ; Zinc finger proteins ; Zinc Fingers - physiology
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descriptionRealizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
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titleEfficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases
authorAmora, Ranier ; Miller, Jeffrey C ; Mitalipova, Maisam ; Katibah, George E ; Rebar, Edward J ; Urnov, Fyodor D ; Jaenisch, Rudolf ; Gregory, Philip D ; Boydston, Elizabeth A ; Beard, Caroline ; Hockemeyer, Dirk ; Zeitler, Bryan ; Meng, Xiangdong ; Soldner, Frank ; Gao, Qing ; Zhang, Lei ; DeKelver, Russell C
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7Fundamental and applied biological sciences. Psychology
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abstractRealizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
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