Efficient generation of conditional knockout mice via sequential introduction of lox sites

Abstract Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites o...

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Autores principales:	Takuro Horii, Sumiyo Morita, Mika Kimura, Naomi Terawaki, Mihiro Shibutani, Izuho Hatada
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b4fd81cd03ed4bcda6d795605d1f2aec
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spelling	oai:doaj.org-article:b4fd81cd03ed4bcda6d795605d1f2aec2021-12-02T16:07:04ZEfficient generation of conditional knockout mice via sequential introduction of lox sites10.1038/s41598-017-08496-82045-2322https://doaj.org/article/b4fd81cd03ed4bcda6d795605d1f2aec2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08496-8https://doaj.org/toc/2045-2322Abstract Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.Takuro HoriiSumiyo MoritaMika KimuraNaomi TerawakiMihiro ShibutaniIzuho HatadaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Takuro Horii Sumiyo Morita Mika Kimura Naomi Terawaki Mihiro Shibutani Izuho Hatada Efficient generation of conditional knockout mice via sequential introduction of lox sites
description	Abstract Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.
format	article
author	Takuro Horii Sumiyo Morita Mika Kimura Naomi Terawaki Mihiro Shibutani Izuho Hatada
author_facet	Takuro Horii Sumiyo Morita Mika Kimura Naomi Terawaki Mihiro Shibutani Izuho Hatada
author_sort	Takuro Horii
title	Efficient generation of conditional knockout mice via sequential introduction of lox sites
title_short	Efficient generation of conditional knockout mice via sequential introduction of lox sites
title_full	Efficient generation of conditional knockout mice via sequential introduction of lox sites
title_fullStr	Efficient generation of conditional knockout mice via sequential introduction of lox sites
title_full_unstemmed	Efficient generation of conditional knockout mice via sequential introduction of lox sites
title_sort	efficient generation of conditional knockout mice via sequential introduction of lox sites
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/b4fd81cd03ed4bcda6d795605d1f2aec
work_keys_str_mv	AT takurohorii efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites AT sumiyomorita efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites AT mikakimura efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites AT naomiterawaki efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites AT mihiroshibutani efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites AT izuhohatada efficientgenerationofconditionalknockoutmiceviasequentialintroductionofloxsites
_version_	1718384800297910272

Efficient generation of conditional knockout mice via sequential introduction of lox sites

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