PMID- 17228519
OWN - NLM
STAT- MEDLINE
DCOM- 20070215
LR  - 20181201
IS  - 1359-4117 (Print)
IS  - 1359-4117 (Linking)
VI  - 6
IP  - 1
DP  - 2006
TI  - Genetic polymorphisms of human ABC transporter ABCG2: development of the standard 
      method for functional validation of SNPs by using the Flp recombinase system.
PG  - 1-11
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. However, integration of cDNA into the 
      host's chromosomal DNA occurs randomly at unpredictable sites, and the number of 
      integrated recombinant DNAs is not controllable. To investigate the effect of 
      genetic polymorphisms of ABCG2 on the protein expression and the drug resistance 
      profile, we developed the Flp-In method to integrate one single copy of ABCG2 
      variant-cDNA into FRT-tagged genomic DNA. More than 20 metaphase spreads were 
      examined for both fluorescence in situ hybridization (FISH) mapping and 
      multicolor-FISH analysis, and it has been revealed that ABCG2 cDNA was 
      incorporated into the telomeric region of the short arm on one of chromosomes 12 
      in Flp-In-293 cells. Based on the currently available SNP data for human ABCG2, 
      we have created a total of seven variants by site-directed mutagenesis and stably 
      expressed them in Flp-In-293 cells. While mRNAs of those integrated ABCG2 
      variants and wild type were evenly expressed in Flp-In-293 cells, the protein 
      expression levels of F208S and S441N variants were found to be markedly low. It 
      is suggested that the protein instability due to enhanced degradation resulted in 
      the low levels of their protein expression. Thus, the Flp recombinase system 
      would provide a useful tool to validate the effect of nonsynonymous SNPs on the 
      protein stability and post-translational modification of ABCG2.
FAU - Tamura, Ai
AU  - Tamura A
AD  - Department of Biomolecular Engineering, Graduate School of Bioscience and 
      Biotechnology, Tokyo Institute of Technology, 4259-B-60 Nagatsuta, Midori-ku, 
      Yokohama 226-8501, Japan.
FAU - Wakabayashi, Kanako
AU  - Wakabayashi K
FAU - Onishi, Yuko
AU  - Onishi Y
FAU - Nakagawa, Hiroshi
AU  - Nakagawa H
FAU - Tsuji, Masahisa
AU  - Tsuji M
FAU - Matsuda, Yoichi
AU  - Matsuda Y
FAU - Ishikawa, Toshihisa
AU  - Ishikawa T
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Exp Ther Oncol
JT  - Journal of experimental therapeutics & oncology
JID - 9604933
RN  - 0 (ABCG2 protein, human)
RN  - 0 (ATP Binding Cassette Transporter, Subfamily G, Member 2)
RN  - 0 (ATP-Binding Cassette Transporters)
RN  - 0 (Calcium Phosphates)
RN  - 0 (DNA, Complementary)
RN  - 0 (Neoplasm Proteins)
RN  - 97Z1WI3NDX (calcium phosphate)
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  - Calcium Phosphates/metabolism
MH  - Chromosome Mapping
MH  - DNA Nucleotidyltransferases/*metabolism
MH  - DNA, Complementary/metabolism
MH  - Drug Resistance, Neoplasm/genetics
MH  - Genetic Vectors
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Metaphase
MH  - Mutagenesis, Site-Directed
MH  - *Mutation
MH  - Neoplasm Proteins/*genetics
MH  - *Polymorphism, Genetic
MH  - *Polymorphism, Single Nucleotide
MH  - Protein Processing, Post-Translational
EDAT- 2007/01/19 09:00
MHDA- 2007/02/16 09:00
CRDT- 2007/01/19 09:00
PHST- 2007/01/19 09:00 [pubmed]
PHST- 2007/02/16 09:00 [medline]
PHST- 2007/01/19 09:00 [entrez]
PST - ppublish
SO  - J Exp Ther Oncol. 2006;6(1):1-11.