A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins |
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Authors: | Takayuki Iwaki Francis J Castellino |
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Institution: | (1) W.M. Keck Center for Transgene Research, University of Notre Dame, 235 Raclin-Carmichael Hall, Notre Dame, IN 46556, USA;(2) Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA |
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Abstract: | The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively. This
system can establish both transient and stable transformants with various selection markers. The generation of stable cell
lines for increased expression or large scale expression of the desired protein is currently accomplished by cotransfection
of both the expression and selection vectors. The selection vectors, pCoHYGRO and pCoBLAST, are commercially available using
hygromycin-B and blasticidin S, respectively. Recently, we generated a plasmid, pCoPURO, for selection of transfected S2 cells
using puromycin, which allows significant acceleration of the selection time. Although co-transfection of the selection marker
with the plasmid for heterologous protein expression is functional in stable expression at short culture periods, the expression
levels of stable transformants are continuously decreased during long culture times. To overcome this limitation, we generated
pMT-PURO, a new plasmid that contains both the expression cassette and puromycin selection marker in a single plasmid. This
system allows rapid selection and maintenance of the transformed S2 lines for extended culture periods. |
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Keywords: | S2 cell Puromycin Selection Single plasmid |
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