PMID- 10512203
OWN - NLM
STAT- MEDLINE
DCOM- 20000124
LR  - 20061115
IS  - 0145-5680 (Print)
IS  - 0145-5680 (Linking)
VI  - 45
IP  - 5
DP  - 1999 Jul
TI  - Exchangeable gene trap using the Cre/mutated lox system.
PG  - 737-50
AB  - The gene trap technique is a powerful approach for characterizing and mutating
      genes involved in mouse development. However, one shortcoming of gene trapping is
      the relative inability to induce subtle mutations. This problem can be overcome
      by introducing a knock-in system into the gene trap strategy. Here, we have
      constructed a new gene trap vector, pU-Hachi, employing the Cre-mutated lox
      system (Araki et al., 1997), in which a pair of mutant lox, lox71 and lox66, was 
      used to promote targeted integrative reaction by Cre recombinase. The pU-Hachi
      carries splicing acceptor (SA)-lox71-internal ribosomal entry site
      (IRES)-beta-geo-pA-loxP-pA-pUC. By using this vector, we can carry out random
      insertional mutagenesis as the first step, and then we can replace the beta-geo
      gene with any gene of interest through Cre-mediated integration. We have isolated
      109 trap clones electroporated with pU-Hachi, and analyzed their integration
      patterns by Southern blotting to select those carrying a single copy of the trap 
      vector. By use of some of these clones, we have succeeded in exchanging the
      reporter gene at high efficiency, ranging between 20-80%. This integration system
      is also quite useful for plasmid rescue to recover flanking genomic sequences,
      because a plasmid vector sequence can be introduced even when the pUC sequence of
      the trap vector is lost through integration into the genome. Thus, this method,
      termed exchangeable gene trapping, has many advantages as the trapped clones can 
      be utilized to express genes with any type of mutation.
FAU - Araki, K
AU  - Araki K
AD  - Institute of Molecular Embryology and Genetics, Kumamoto University School of
      Medicine, Japan. yamamura@gpo.kumamoto-u.ac.jp
FAU - Imaizumi, T
AU  - Imaizumi T
FAU - Sekimoto, T
AU  - Sekimoto T
FAU - Yoshinobu, K
AU  - Yoshinobu K
FAU - Yoshimuta, J
AU  - Yoshimuta J
FAU - Akizuki, M
AU  - Akizuki M
FAU - Miura, K
AU  - Miura K
FAU - Araki, M
AU  - Araki M
FAU - Yamamura, K
AU  - Yamamura K
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - France
TA  - Cell Mol Biol (Noisy-le-grand)
JT  - Cellular and molecular biology (Noisy-le-Grand, France)
JID - 9216789
RN  - 0 (Luminescent Proteins)
RN  - 0 (Viral Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 2.7.7.- (Cre recombinase)
RN  - EC 2.7.7.- (Integrases)
RN  - EC 3.2.1.23 (beta-Galactosidase)
SB  - IM
MH  - Animals
MH  - Clone Cells
MH  - *Genetic Techniques
MH  - *Genetic Vectors
MH  - Green Fluorescent Proteins
MH  - Integrases/*genetics
MH  - Luminescent Proteins/genetics
MH  - Mice
MH  - Mutagenesis
MH  - Mutation
MH  - Plasmids/genetics
MH  - Stem Cells
MH  - *Viral Proteins
MH  - beta-Galactosidase/genetics
EDAT- 1999/10/08 00:00
MHDA- 1999/10/08 00:01
CRDT- 1999/10/08 00:00
PHST- 1999/10/08 00:00 [pubmed]
PHST- 1999/10/08 00:01 [medline]
PHST- 1999/10/08 00:00 [entrez]
PST - ppublish
SO  - Cell Mol Biol (Noisy-le-grand). 1999 Jul;45(5):737-50.