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

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

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Article number274
JournalBMC genomics
Volume16
Issue number1
DOIs
StatePublished - Apr 9 2015

Fingerprint

Gene Transfer Techniques
Transgenic Mice
Injections
Transgenes
Zygote
Genome
Technology

Keywords

  • Cre-loxP
  • FLP-FRT
  • PhiC31-attP/B
  • Pronuclear injection-based targeted transgenesis
  • Rosa26
  • Transgenic mouse
  • Transportation of the cauda epididymides

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

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.

In: BMC genomics, Vol. 16, No. 1, 274, 09.04.2015.

Research output: Contribution to journalArticle

Ohtsuka, M, Miura, H, Mochida, K, Hirose, M, Hasegawa, A, Ogura, A, Mizutani, R, Kimura, M, Isotani, A, Ikawa, M, Sato, M & Gurumurthy, CB 2015, 'One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)', BMC genomics, vol. 16, no. 1, 274. https://doi.org/10.1186/s12864-015-1432-5
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. / One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). In: BMC genomics. 2015 ; Vol. 16, No. 1.
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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.",
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AU - Mochida, Keiji

AU - Hirose, Michiko

AU - Hasegawa, Ayumi

AU - Ogura, Atsuo

AU - Mizutani, Ryuta

AU - Kimura, Minoru

AU - Isotani, Ayako

AU - Ikawa, Masahito

AU - Sato, Masahiro

AU - Gurumurthy, Channabasavaiah B

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AB - 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.

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