Selectable marker gene bsrm induces death of keratinocytes
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2002
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ANNUAL MEETING OF THE AMARICAN SOCIETY OF GENE THERAPY, 5th
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Resumo
Gene transfer into cultured mammalian cells and the identification of transformed cells with a
selectable marker are common steps in gene transfer and gene therapy protocols. The blasticidin S
resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, is used as a dominant selectable
marker in gene transfer to mammalian cells. Recently, we reported a construction of improved form
of bsr (bsrm), which provided higher protection against BS in the cells transduced with that gene by
retroviral vector. Keratinocytes are powerful target cells to ex vivo gene therapy protocols, although
the selection process still needs to be improved due to the extreme sensitivity of these cells to drugs.
Therefore, we decided to use the bsrm gene as a selectable marker to transfer therapeutic genes into
the mouse keratinocytes BALB/MK cells. Surprisingly, the transduced cells died within 6 days of
culture. Cell cycle analysis by flow cytometry on the 6th day demonstrated that the majority of cells
were in the sub-GO phase. A ring-shaped condensed chromatin was also observed upon staining of
bsrm-transduced BALB/MK cells with HOECHST 33258 dye after 6 days of culture. Additionally, the
caspases-3 was also activated on day 6. These findings indicate that BALB/MK transduced with bsrm
gene died by apoptosis. Most importantly, whereas primary human keratinocytes also died in
response to bsrm, rat vascular smooth muscle cells and NIH3T3 mouse fibroblasts were resistant to
the same manipulation. Taken together, our results suggest that the toxic effect of bsrm is somehow
specific for keratinocytes. Therefore, we proposed that even if the bsrm/BS selection system appears
to be powerful to eliminate non-transduced cells efficiently, care should be taken to employ it in
gene transfer experiments, at least the ones involving keratinocytes.
Como referenciar
BENTO, F.M.; MATHOR, M.B.; CHAMMAS, R.; BELIZARIO, J.E.; ULBRICH, A.G.; AMARANTE MENDES, G.P.; HAN, S.W. Selectable marker gene bsrm induces death of keratinocytes. In: ANNUAL MEETING OF THE AMARICAN SOCIETY OF GENE THERAPY, 5th, June 5-9, 2002, Boston, MA, USA. Abstracts... Disponível em: http://repositorio.ipen.br/handle/123456789/20950. Acesso em: 30 Dec 2025.
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