Navegando por Palavras-chave "selectable marker"

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    Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide
    (Natl Research Council Canada-n R C Research Press, 2007-10-01) Takeshita, Daniela; Bento, Fernanda Mara; Chammas, Roger; Belizario, Jose Ernesto; Carmona, Adriana Karaoglanovic; Konno, Katsuhiro; Melo, Robson Lopes de; Molina, Gustavo; Lisboa, Bianca Cristina Garcia; Han, Sang Won [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Butantan Inst
    Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. in this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) the 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of I amino acid. (ii) the conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.
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    Modified blasticidin S resistance gene (bsrm) as a selectable marker for construction of retroviral vectors
    (Elsevier B.V., 2002-04-25) Freitas, A. C.; Bento, F. M.; Ramesh, N.; Osborne, WRA; Han, S. W.; Universidade Federal de São Paulo (UNIFESP); Univ Washington
    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. (C) 2002 Elsevier Science B.V. All rights reserved.