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Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we emplo...

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Autores principales: Ramachandra, Chrishan J. A., Shahbazi, Mohammad, Kwang, Timothy W. X., Choudhury, Yukti, Bak, Xiao Ying, Yang, Jing, Wang, Shu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167641/
https://www.ncbi.nlm.nih.gov/pubmed/21685448
http://dx.doi.org/10.1093/nar/gkr409
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author Ramachandra, Chrishan J. A.
Shahbazi, Mohammad
Kwang, Timothy W. X.
Choudhury, Yukti
Bak, Xiao Ying
Yang, Jing
Wang, Shu
author_facet Ramachandra, Chrishan J. A.
Shahbazi, Mohammad
Kwang, Timothy W. X.
Choudhury, Yukti
Bak, Xiao Ying
Yang, Jing
Wang, Shu
author_sort Ramachandra, Chrishan J. A.
collection PubMed
description Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
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spelling pubmed-31676412011-09-06 Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors Ramachandra, Chrishan J. A. Shahbazi, Mohammad Kwang, Timothy W. X. Choudhury, Yukti Bak, Xiao Ying Yang, Jing Wang, Shu Nucleic Acids Res Methods Online Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome. Oxford University Press 2011-09 2011-06-17 /pmc/articles/PMC3167641/ /pubmed/21685448 http://dx.doi.org/10.1093/nar/gkr409 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Ramachandra, Chrishan J. A.
Shahbazi, Mohammad
Kwang, Timothy W. X.
Choudhury, Yukti
Bak, Xiao Ying
Yang, Jing
Wang, Shu
Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title_full Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title_fullStr Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title_full_unstemmed Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title_short Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
title_sort efficient recombinase-mediated cassette exchange at the aavs1 locus in human embryonic stem cells using baculoviral vectors
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167641/
https://www.ncbi.nlm.nih.gov/pubmed/21685448
http://dx.doi.org/10.1093/nar/gkr409
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