ABCMdb

PMID: 12659681

Ujhelly O, Ozvegy C, Varady G, Cervenak J, Homolya L, Grez M, Scheffer G, Roos D, Bates SE, Varadi A, Sarkadi B, Nemet K
Application of a human multidrug transporter (ABCG2) variant as selectable marker in gene transfer to progenitor cells.
Hum Gene Ther. 2003 Mar 1;14(4):403-12., 2003-03-01 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Arg482Gly In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate specificity, was coexpressed with a therapeutic gene by using a bicistronic vector and an efficient retroviraltransduction protocol. Login to comment 17 ABCG2 p.Arg482Gly ABCG2 is normally expressed in the placenta and in stem cells (Zhou et al., 2001; Bunting,2002; Kim et al., 2002).It has been shown that the R482G variant of ABCG2 has a different substrate specificity than the wild-type protein (Honjo et al., 2001; Öz- 1National Medical Center, Institute of Hematology and Immunology, 1113 Budapest, Hungary. Login to comment 37 ABCG2 p.Arg482Gly The NotI-BamHI fragment from the coding sequence of the R482G mutant MXR cDNA was used to replace the previously used LNGFR cDNA. Login to comment 39 ABCG2 p.Arg482Gly In a monocistronicABCG2 vector, LNGFR cDNA was replaced by the cDNAs encoding the R482 (SsARS) and R482G (SsAGS) variants of ABCG2, and the gp91phox cDNA was also removed. Login to comment 82 ABCG2 p.Arg482Gly RESULTS AND DISCUSSION Coexpression of gp91phox and ABCG2 in progenitor cells: effect of drug selection To produce recombinant retroviruses,a bicistronic retroviral vector was constructed, carrying the coding sequences for gp91phox and those for the normal ABCG2, or the R482G variant (denoted as ABCG2-G). Login to comment 145 ABCG2 p.Arg482Gly Selective protection of the transduced cells against cytotoxic drugs by the mutant ABCG2 protein In the above-describedexperiments we used an R482G variant of the ABCG2 protein as a selectable marker. Login to comment 147 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly It has been reported that the R482G and R482T mutant variants of ABCG2 have altered substrate specificity. Login to comment 149 ABCG2 p.Arg482Gly To investigate the function and expression of these variants, we generated cDNAs encoding both the wild-type and R482G ABCG2 proteins, and inserted each of these cDNAs into the retroviral vector presented above (see Fig. 1). Login to comment 153 ABCG2 p.Arg482Gly As documented in Fig. 5, after retroviral transduction and selection for 4 days in increasing concentrations of MX in the medium, PLB985 cells expressed both the wild-type ABCG2 and the R482G variant of ABCG2 in a functionallyactive form. Login to comment 176 ABCG2 p.Arg482Gly of Western blot lanes (using actin as an internal protein control), PLB985 cells transduced with wild-type ABCG2 and selected in 10 nM MX (Fig. 5A, lane 4) expressed a similar (according to PhosphorImager evaluation, 1.6 times higher) level of ABCG2 protein than did corresponding cells expressing the R482G variant and selected in 100 nM MX (Fig. 5A, lane 5). Login to comment 188 ABCG2 p.Arg482Gly In summary, we have demonstrated that overexpression of R482G ABCG2 protein efficiently protected genetically modified progenitor cells against cytotoxic selection, allowing increased expression of the therapeutic gene. Login to comment