Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system.
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.
Tamura
Ai
A
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-60 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Wakabayashi
Kanako
K
Onishi
Yuko
Y
Nakagawa
Hiroshi
H
Tsuji
Masahisa
M
Matsuda
Yoichi
Y
Ishikawa
Toshihisa
T
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D002130:Calcium Phosphates / Q000378:metabolism; D002874:Chromosome Mapping; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011499:Protein Processing, Post-Translational
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002130:Calcium Phosphates; D018076:DNA, Complementary; D009363:Neoplasm Proteins; C020243:calcium phosphate; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase