Další formáty:
BibTeX
LaTeX
RIS
@article{1395493, author = {Bohačiaková, Dáša and Renzová, Tereza and Fedorová, Veronika and Barák, Martin and Bosáková, Michaela and Hampl, Aleš and Čajánek, Lukáš}, article_location = {New York}, article_number = {21}, doi = {http://dx.doi.org/10.1089/scd.2017.0058}, keywords = {human embryonic stem cells; CRISPR/Cas9; gene engineering; knockout}, language = {eng}, issn = {1547-3287}, journal = {Stem Cells and Development}, title = {An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System}, url = {http://online.liebertpub.com/doi/10.1089/scd.2017.0058}, volume = {26}, year = {2017} }
TY - JOUR ID - 1395493 AU - Bohačiaková, Dáša - Renzová, Tereza - Fedorová, Veronika - Barák, Martin - Bosáková, Michaela - Hampl, Aleš - Čajánek, Lukáš PY - 2017 TI - An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System JF - Stem Cells and Development VL - 26 IS - 21 SP - 1521-1527 EP - 1521-1527 PB - Mary Ann Liebert, Inc. SN - 15473287 KW - human embryonic stem cells KW - CRISPR/Cas9 KW - gene engineering KW - knockout UR - http://online.liebertpub.com/doi/10.1089/scd.2017.0058 N2 - Human embryonic stem cells (hESCs) represent a 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/CRISPR-associated protein-9 nuclease (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 a 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 hESCs 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 ER -
BOHAČIAKOVÁ, Dáša, Tereza RENZOVÁ, Veronika FEDOROVÁ, Martin BARÁK, Michaela BOSÁKOVÁ, Aleš HAMPL a Lukáš ČAJÁNEK. An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System. \textit{Stem Cells and Development}. New York: Mary Ann Liebert, Inc., 2017, roč.~26, č.~21, s.~1521-1527. ISSN~1547-3287. Dostupné z: https://dx.doi.org/10.1089/scd.2017.0058.
|