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Selective gene silencing of rat ATP‐binding cassette G2 transporter in an in vitro blood–brain barrier model by short interfering RNA
Author(s) -
Hori Satoko,
Ohtsuki Sumio,
Ichinowatari Masashi,
Yokota Takanori,
Kanda Takashi,
Terasaki Tetsuya
Publication year - 2005
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2004.02994.x
Subject(s) - small interfering rna , transfection , microbiology and biotechnology , hek 293 cells , gene silencing , blood–brain barrier , biology , gene knockdown , rna interference , messenger rna , rna , western blot , gene expression , gene , biochemistry , neuroscience , central nervous system
The aim of the present study was to specifically silence the rat ATP‐binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc‐tagged rABCG2 cDNA. Quantitative real‐time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2‐mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non‐specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR‐BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR‐BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood–brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain‐to‐blood transport of substrate drugs and endogenous compounds across the blood–brain barrier.