PMID- 20700710
OWN - NLM
STAT- MEDLINE
DCOM- 20101122
LR  - 20181201
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 648
DP  - 2010
TI  - Production of cells with targeted integration of gene variants of human ABC 
      transporter for stable and regulated expression using the Flp recombinase system.
PG  - 139-59
LID - 10.1007/978-1-60761-756-3_9 [doi]
AB  - The vector-mediated introduction of cDNA into mammalian cells by calcium 
      phosphate co-precipitation or permeation with lipofectamine is widely used for 
      the integration of cDNA into genomic DNA. Such integration, however, of cDNA 
      occurs randomly at unpredictable sites in the host's chromosomal DNA, and the 
      number of integrated recombinant DNAs is not controllable. To overcome this 
      problem, we developed the Flp-In method to integrate one single copy of cDNA 
      encoding the human ABC transporter ABCG2 into FRT-tagged genomic DNA. Examination 
      of more than 20 metaphase spreads for both fluorescence in situ hybridization 
      (FISH) mapping and multicolor-FISH analysis revealed that ABCG2 cDNA was 
      incorporated into the telomeric region of the short arm on one of chromosomes 12 
      in Flp-In-293 cells. By using those cells, we investigated the effect of genetic 
      polymorphisms and post-translational modifications of human ABC transporter ABCG2 
      on the protein expression and degradation. On the basis of our experience, it has 
      been concluded that the Flp recombinase system provides a useful tool to 
      quantitatively analyze the protein stability and endoplasmic reticulum 
      (ER)-associated degradation of proteins like the ABC transporter. This system is 
      also applicable for similar studies of the biogenesis of other proteins using the 
      secretory pathway as well as proteins with other cellular localizations.
FAU - Wakabayashi-Nakao, Kanako
AU  - Wakabayashi-Nakao K
FAU - Tamura, Ai
AU  - Tamura A
FAU - Koshiba, Shoko
AU  - Koshiba S
FAU - Toyoda, Yu
AU  - Toyoda Y
FAU - Nakagawa, Hiroshi
AU  - Nakagawa H
FAU - Ishikawa, Toshihisa
AU  - Ishikawa T
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (ABCG2 protein, human)
RN  - 0 (ATP Binding Cassette Transporter, Subfamily G, Member 2)
RN  - 0 (ATP-Binding Cassette Transporters)
RN  - 0 (DNA, Complementary)
RN  - 0 (Neoplasm Proteins)
RN  - EC 2.7.7.- (DNA Nucleotidyltransferases)
RN  - EC 2.7.7.- (FLP recombinase)
SB  - IM
MH  - ATP Binding Cassette Transporter, Subfamily G, Member 2
MH  - ATP-Binding Cassette Transporters/*genetics
MH  - Animals
MH  - Base Sequence
MH  - Cell Line
MH  - DNA Nucleotidyltransferases/*metabolism
MH  - DNA, Complementary/genetics
MH  - *Gene Expression
MH  - Gene Targeting/*methods
MH  - Humans
MH  - Immunoblotting
MH  - Molecular Sequence Data
MH  - Mutagenesis, Insertional/*genetics
MH  - Mutation/*genetics
MH  - Neoplasm Proteins/*genetics
MH  - Reproducibility of Results
EDAT- 2010/08/12 06:00
MHDA- 2010/12/14 06:00
CRDT- 2010/08/12 06:00
PHST- 2010/08/12 06:00 [entrez]
PHST- 2010/08/12 06:00 [pubmed]
PHST- 2010/12/14 06:00 [medline]
AID - 10.1007/978-1-60761-756-3_9 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2010;648:139-59. doi: 10.1007/978-1-60761-756-3_9.