title: One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).
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.
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. hiromi@tokai-u.jp.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. jmochida@rtc.riken.jp.
authors.forename: Keiji
authors.initials: K
authors.lastname: Mochida
authors.affiliation: RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. m-hirose@rtc.riken.jp.
authors.forename: Michiko
authors.initials: M
authors.lastname: Hirose
authors.affiliation: RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. a-hasegawa@rtc.riken.jp.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Hasegawa
authors.affiliation: RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. ogura@rtc.riken.jp.|Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki, 305-8572, Japan. ogura@rtc.riken.jp.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.affiliation: Graduate School of Engineering, Tokai University, Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan. ryuta@tokai-u.jp.
authors.forename: Ryuta
authors.initials: R
authors.lastname: Mizutani
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. kimura@is.icc.u-tokai.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. isotani@biken.osaka-u.ac.jp.
authors.forename: Ayako
authors.initials: A
authors.lastname: Isotani
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. ikawa@biken.osaka-u.ac.jp.
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima, 890-8544, Japan. masasato@ms.kagoshima-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA. cgurumurthy@unmc.edu.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells; D005260:Female; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D007267:Injections / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic