An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing

Abstract Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Fu...

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Autores principales:	Ako Eugene Ako, Pierre-François Perroud, Joseph Innocent, Viktor Demko, Odd-Arne Olsen, Wenche Johansen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5d791c27f43f41b2865c04cd62b2a9bd
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spelling	oai:doaj.org-article:5d791c27f43f41b2865c04cd62b2a9bd2021-12-02T15:05:54ZAn intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing10.1038/s41598-017-05309-w2045-2322https://doaj.org/article/5d791c27f43f41b2865c04cd62b2a9bd2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-05309-whttps://doaj.org/toc/2045-2322Abstract Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5′ end of the intron and preserve the nucleotide sequence of the targeted intron.Ako Eugene AkoPierre-François PerroudJoseph InnocentViktor DemkoOdd-Arne OlsenWenche JohansenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ako Eugene Ako Pierre-François Perroud Joseph Innocent Viktor Demko Odd-Arne Olsen Wenche Johansen An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
description	Abstract Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5′ end of the intron and preserve the nucleotide sequence of the targeted intron.
format	article
author	Ako Eugene Ako Pierre-François Perroud Joseph Innocent Viktor Demko Odd-Arne Olsen Wenche Johansen
author_facet	Ako Eugene Ako Pierre-François Perroud Joseph Innocent Viktor Demko Odd-Arne Olsen Wenche Johansen
author_sort	Ako Eugene Ako
title	An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
title_short	An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
title_full	An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
title_fullStr	An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
title_full_unstemmed	An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing
title_sort	intragenic mutagenesis strategy in physcomitrella patens to preserve intron splicing
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/5d791c27f43f41b2865c04cd62b2a9bd
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An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing

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