One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Ohtsuka, Masato; Miura, Hiromi; Mochida, Keiji; Hirose, Michiko; Hasegawa, Ayumi; Ogura, Atsuo; Mizutani, Ryuta; Kimura, Minoru; Isotani, Ayako; Ikawa, Masahito; Sato, Masahiro; Gurumurthy, Channabasavaiah B.

doi:https://doi.org/10.1186/s12864-015-1432-5

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Ohtsuka, Masato; Miura, Hiromi; Mochida, Keiji et al.

BMC Genomics, 2015, 16(1), 274

アクセス数:252件（2025-04-18 02:27 集計）

固定URL: https://hdl.handle.net/11094/78599

閲覧可能ファイル

ファイル	フォーマット	利用条件	サイズ	閲覧回数	利用開始日	説明	information
BMCGenomics_16_01_274	pdf	なし	2.40 MB	144	2021.02.08

Creative Commons : 表示

論文情報

ファイル出力 EndNote Basic出力 Mendeley出力

タイトル	One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)
著者	NRID 1000090372945 1000090372945 著者名 Ohtsuka, Masato
	NRID 1000090599523 1000090599523 著者名 Miura, Hiromi
	NRID 1000060312287 1000060312287 著者名 Mochida, Keiji
	著者名 Hirose, Michiko
	著者名 Hasegawa, Ayumi
	NRID 1000020194524 1000020194524 著者名 Ogura, Atsuo
	NRID 1000070272482 1000070272482 著者名 Mizutani, Ryuta
	NRID 1000010146706 1000010146706 著者名 Kimura, Minoru
	NRID 1000020444523 1000020444523 著者名 Isotani, Ayako
	NRID 1000020304066 1000020304066 ORCID 0000-0001-9859-6217 0000-0001-9859-6217 researchmap permalink read0054910 read0054910 Scopus Author ID 16689054500 16689054500 著者名 Ikawa, Masahito
	NRID 1000030287099 1000030287099 著者名 Sato, Masahiro
	著者名 Gurumurthy, Channabasavaiah B.
キーワード等	Cre-loxP
	FLP-FRT
	PhiC31-attP/B
	Pronuclear injection-based targeted transgenesis
	Rosa26
	Transgenic mouse
	Transportation of the cauda epididymides
内容	Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-5
抄録	Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. Results: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.
出版者	BioMed Central Ltd.
収録物名	BMC Genomics
巻(号)	16(1)
ページ	274
刊行年月	2015-04-09
言語	英語
ハンドルURL	https://hdl.handle.net/11094/78599
eISSN	14712164
NCID	AA12034967
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1186/s12864-015-1432-5
関連情報 (isIdenticalTo)	PMID 25887549
アクセス権	open access
権利情報	© 2015 Ohtsuka et al.; licensee BioMed Central. This article is licensed under a Creative Commons Attribution 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCTERMS.bibliographicCitation	BMC Genomics. 2015, 16(1), p. 274
DC.title	One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)
DC.creator	Ohtsuka, Masato
	Miura, Hiromi
	Mochida, Keiji
	Hirose, Michiko
	Hasegawa, Ayumi
	Ogura, Atsuo
	Mizutani, Ryuta
	Kimura, Minoru
	Isotani, Ayako
	Ikawa, Masahito
	Sato, Masahiro
	Gurumurthy, Channabasavaiah B.
DC.publisher	BioMed Central Ltd.
DC.language' scheme='DCTERMS.RFC1766	英語
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2015-04-09
DC.identifier	https://doi.org/10.1186/s12864-015-1432-5
DC.subject	Cre-loxP
	FLP-FRT
	PhiC31-attP/B
	Pronuclear injection-based targeted transgenesis
	Rosa26
	Transgenic mouse
	Transportation of the cauda epididymides
DC.description	Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-5
DCTERMS.abstract	Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. Results: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.
DC.rights	© 2015 Ohtsuka et al.; licensee BioMed Central. This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	78599
citation_title	One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)
citation_author	Ohtsuka, Masato
	Miura, Hiromi
	Mochida, Keiji
	Hirose, Michiko
	Hasegawa, Ayumi
	Ogura, Atsuo
	Mizutani, Ryuta
	Kimura, Minoru
	Isotani, Ayako
	Ikawa, Masahito
	Sato, Masahiro
	Gurumurthy, Channabasavaiah B.
citation_publisher	BioMed Central Ltd.
citation_language	英語
citation_date	2015-04-09
citation_journal_title	BMC Genomics
citation_volume	16
citation_issue	1
citation_firstpage	274
citation_doi	https://doi.org/10.1186/s12864-015-1432-5
citation_pmid	25887549
citation_public_url	https://hdl.handle.net/11094/78599

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

閲覧可能ファイル

論文情報

論文詳細を表示