An efficient method for generation of Knockout human embryonic stem cells using CRISPR/Cas9 system
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F17%3A00067189" target="_blank" >RIV/00159816:_____/17:00067189 - isvavai.cz</a>
Alternative codes found
RIV/00216224:14110/17:00095168
Result on the web
<a href="http://dx.doi.org/10.1089/scd.2017.0058" target="_blank" >http://dx.doi.org/10.1089/scd.2017.0058</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1089/scd.2017.0058" target="_blank" >10.1089/scd.2017.0058</a>
Alternative languages
Result language
angličtina
Original language name
An efficient method for generation of Knockout human embryonic stem cells using CRISPR/Cas9 system
Original language description
Human embryonic stem cells (hESCs) represent promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/Cas9 system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESC do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
30205 - Hematology
Result continuities
Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)
Others
Publication year
2017
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Stem Cells and Development
ISSN
1547-3287
e-ISSN
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Volume of the periodical
26
Issue of the periodical within the volume
21
Country of publishing house
US - UNITED STATES
Number of pages
7
Pages from-to
1521-1527
UT code for WoS article
000413639900001
EID of the result in the Scopus database
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