Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression.
Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.
Ohtsuka
Masato
M
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
Ogiwara
Sanae
S
Miura
Hiromi
H
Mizutani
Akiko
A
Warita
Takayuki
T
Sato
Masahiro
M
Imai
Kenji
K
Hozumi
Katsuto
K
Sato
Takehito
T
Tanaka
Masafumi
M
Kimura
Minoru
M
Inoko
Hidetoshi
H
D000818:Animals; D002467:Cell Nucleus; D053595:Embryonic Stem Cells / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018390:Gene Targeting / Q000379:methods*; D007267:Injections; D019426:Integrases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010063:Ovum / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion; D019076:Transgenes
C045073:Cre recombinase; D019426:Integrases