GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives

Autor: Elizabeth L. Qian, Tanya Labonne, Koula Sourris, Freya Bruveris, Ritu Chaurasia, Arash Rafii, David A. Anderson, Jacqueline V. Schiesser, Tim Kao, Andrew G. Elefanty, Zerina Lokmic, Clare L. Parish, Michael Cheung, Shireen R. Lamandé, Ali Motazedian, Antonietta Giudice, Magdaline Costa, Lachlan H. Thomson, Sara E. Howden, Peter G. Farlie, Elizabeth Ng, Anthony J. Penington, Edouard G. Stanley, David A. Elliott, Jonathan C. Niclis
Přispěvatelé: Murdoch Children’s Research Institute [Melbourne, Australia], University of Melbourne, The Florey Institute of Neuroscience and Mental Health [Parkville, VIC, Australie], Monash University [Clayton], Cincinnati Children's Hospital Medical Center, Monash University [Melbourne], The Royal Children's Hospital, Melbourne, Australia, Développement embryonnaire précoce humain et pluripotence EmbryoPluripotency (UMR 1203), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-CHU Montpellier, Weill Cornell Medical College in Qatar (WCMC-Q), Weill Cornell Medicine [Qatar], Herrada, Anthony
Jazyk: angličtina
Rok vydání: 2016
Předmět:
0301 basic medicine
MESH: CRISPR-Cas Systems
Cellular differentiation
Gene Expression
[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC]
Biochemistry
Green fluorescent protein
expression system
Genes
Reporter
MESH: Genetic Vectors
Clustered Regularly Interspaced Short Palindromic Repeats
Transgenes
human pluripotent stem cells
Induced pluripotent stem cell
MESH: Transcription Activator-Like Effector Nucleases
lcsh:QH301-705.5
lcsh:R5-920
GAPDH
Cell Differentiation
differentiation
medicine.anatomical_structure
[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry
Molecular Biology/Genomics [q-bio.GN]
MESH: Pluripotent Stem Cells
lcsh:Medicine (General)
Pluripotent Stem Cells
Resource
MESH: Cell Differentiation
Mesoderm
Cell type
MESH: Gene Expression
Genetic Vectors
MESH: Transgenes
Germ layer
Biology
Cell Line
03 medical and health sciences
lineage tracing
Transcription Activator-Like Effector Nucleases
[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry
Molecular Biology/Genomics [q-bio.GN]
Genetics
medicine
[SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC]
Humans
Reporter gene
MESH: Humans
MESH: Genes
Reporter
Cell Biology
Molecular biology
MESH: Cell Line
030104 developmental biology
lcsh:Biology (General)
MESH: Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
reporter genes
mCherry
Developmental Biology
Zdroj: Current Stem Cell Reports
Current Stem Cell Reports, Springer, 2016, 7 (3), pp.518-526. ⟨10.1016/j.stemcr.2016.07.015⟩
Stem Cell Reports, Vol 7, Iss 3, Pp 518-526 (2016)
Stem Cell Reports
Web of Science
ISSN: 2198-7866
Popis: Summary The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
Graphical Abstract Image 1
Highlights • GAPTrap vector system targets transgenes to the ubiquitously expressed GAPDH locus • Targeting transgenes to the GAPDH locus yields reliable transgene expression • Transgenes at this locus are robustly expressed in differentiated cells • Generation of GAPTrap targeted human PSC lines is simple and efficient
In this article Stanley, Elefanty, and colleagues describe a simple vector system, GAPTrap, for constitutively expressing transgenes in pluripotent stem cells and their differentiated derivatives. GAPTrap vectors target transgenes to the GAPDH locus, ensuring robust and reliable expression in a wide variety of cell types, and providing an ideal base for cell tagging and gene overexpression experiments.
Databáze: OpenAIRE
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