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Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology
The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132024/ https://www.ncbi.nlm.nih.gov/pubmed/25118822 http://dx.doi.org/10.1371/journal.pone.0104816 |
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author | Porro, Fabiola Bockor, Luka De Caneva, Alessia Bortolussi, Giulia Muro, Andrés F. |
author_facet | Porro, Fabiola Bockor, Luka De Caneva, Alessia Bortolussi, Giulia Muro, Andrés F. |
author_sort | Porro, Fabiola |
collection | PubMed |
description | The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI. |
format | Online Article Text |
id | pubmed-4132024 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-41320242014-08-19 Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology Porro, Fabiola Bockor, Luka De Caneva, Alessia Bortolussi, Giulia Muro, Andrés F. PLoS One Research Article The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI. Public Library of Science 2014-08-13 /pmc/articles/PMC4132024/ /pubmed/25118822 http://dx.doi.org/10.1371/journal.pone.0104816 Text en © 2014 Porro 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 Porro, Fabiola Bockor, Luka De Caneva, Alessia Bortolussi, Giulia Muro, Andrés F. Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title | Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title_full | Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title_fullStr | Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title_full_unstemmed | Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title_short | Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology |
title_sort | generation of ugt1-deficient murine liver cell lines using talen technology |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132024/ https://www.ncbi.nlm.nih.gov/pubmed/25118822 http://dx.doi.org/10.1371/journal.pone.0104816 |
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