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A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells
Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) inc...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661507/ https://www.ncbi.nlm.nih.gov/pubmed/23717601 http://dx.doi.org/10.1371/journal.pone.0064342 |
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author | Chakraborty, Syandan Christoforou, Nicolas Fattahi, Ali Herzog, Roland W. Leong, Kam W. |
author_facet | Chakraborty, Syandan Christoforou, Nicolas Fattahi, Ali Herzog, Roland W. Leong, Kam W. |
author_sort | Chakraborty, Syandan |
collection | PubMed |
description | Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies. |
format | Online Article Text |
id | pubmed-3661507 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-36615072013-05-28 A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells Chakraborty, Syandan Christoforou, Nicolas Fattahi, Ali Herzog, Roland W. Leong, Kam W. PLoS One Research Article Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies. Public Library of Science 2013-05-22 /pmc/articles/PMC3661507/ /pubmed/23717601 http://dx.doi.org/10.1371/journal.pone.0064342 Text en © 2013 Chakraborty et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Chakraborty, Syandan Christoforou, Nicolas Fattahi, Ali Herzog, Roland W. Leong, Kam W. A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title | A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title_full | A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title_fullStr | A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title_full_unstemmed | A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title_short | A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells |
title_sort | robust strategy for negative selection of cre-loxp recombination-based excision of transgenes in induced pluripotent stem cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661507/ https://www.ncbi.nlm.nih.gov/pubmed/23717601 http://dx.doi.org/10.1371/journal.pone.0064342 |
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