Journal of Biotechnology 95 (2002) 57 – 62 Modified blasticidin S resistance gene (bsrm ) as a selectable marker for construction of retroviral vectors Antonio C. Freitas a , Fernanda M. Bento a , Nagarajan Ramesh b , William R.A. Osborne b , Sang W. Han a, * a Department of Biophysics, UNIFESP-EPM, Rua Botucatu 862, Sa ˜o Paulo, SP 04023 -062, Brazil b Department of Pediatrics, Uniersity of Washington, Seattle, WA, USA Received 8 June 2001; received in revised form 26 November 2001; accepted 5 December 2001 Abstract Retroviral vectors are commonly used in ex vivo gene therapy protocols. The structure of vectors basically consists of one gene of interest and a selectable marker gene. Fast selection without damaging cells is a critical step for ex vivo gene therapy protocols. Blasticidin S deaminase isolated from Bacillus cereus has a neutralizing action on the highly toxic antibiotic blasticidin S (BS). A commercially available gene coding for blasticidin S deaminase (bsr ) when used to construct retroviral vectors, LBSN and LNSB, provided very low levels of BS deaminase activity, precluding their routine use in gene transfer experiments. However, with the introduction of specific mutations into the bsr gene based on the Kozak consensus sequences and deletion of a 5 untranslated sequence to generate bsrm, we were able to construct a retroviral vector encoding resistance to high doses of BS (at least 16-fold above the usual lethal dose in NIH3T3 cells), showing that bsrm/BS may provide a useful system for selection of transduced mammalian cells. © 2002 Elsevier Science B.V. All rights reserved. Keywords: bsr; Selectable marker; Retroviral vector; Mutagenesis; Blasticidin S deaminase www.elsevier.com/locate/jbiotec 1. Introduction Gene transfer into cultured mammalian cells and identification of transduced cells with a se- lectable marker are fundamental steps in retrovi- ral-mediated gene therapy protocols (Eglitis, 1991). Since the prokaryotic neomycin phospho- transferase gene (neo) was first described as con- ferring resistance to the antibiotic G418 in yeast (Jimenez and Davies, 1980), the neo/G418 system has found a wide application in a variety of mammalian cells. In a majority of retroviral vec- tors, the neo gene has become the main compo- nent in selection systems (Eglitis et al., 1985; Hock et al., 1989; Miller and Rosman, 1989). However, its use cannot be generalized. For ex- ample, in human keratinocytes, the G418-selected cells had a reduced proliferative potential and altered morphology indicating terminal differenti- ation (Stockschlaeder et al., 1991). For gene ther- * Corresponding author. Tel.: +55-11-5576-4530/5576- 4555x202; fax: +55-11-5571-5780. E-mail address: sang@biofis.epm.br (S.W. Han). 0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0168-1656(01)00442-4