Modified blasticidin S resistance gene (bsrm) as a selectable marker for construction of retroviral vectors.
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.
Freitas
Antonio C
AC
Department of Biophysics, UNIFESP-EPM, Rua Botucatu 862, São Paulo, SP 04023-062, Brazil.
Bento
Fernanda M
FM
Ramesh
Nagarajan
N
Osborne
William R A
WR
Han
Sang W
SW
D016475:3T3 Cells; D000619:Aminohydrolases / Q000235:genetics*; D000818:Animals; D024882:Drug Resistance, Viral / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D009705:Nucleosides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D012641:Selection, Genetic; D014161:Transduction, Genetic / Q000379:methods*
D005819:Genetic Markers; D009705:Nucleosides; D012333:RNA, Messenger; C004500:blasticidin S; D000619:Aminohydrolases; C019795:blasticidin S deaminase