Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.
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September 12 2005
Counterselection and Co-Delivery of Transposon and Transposase Functions for Sleeping Beauty-Mediated Transposition in Cultured Mammalian Cells
Andrea D. Converse;
Andrea D. Converse
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
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Lalitha R. Belur;
Lalitha R. Belur
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
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Jennifer L. Gori;
Jennifer L. Gori
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
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Geyi Liu;
Geyi Liu
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
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Felipe Amaya;
Felipe Amaya
2Advantagene, Inc, San Diego, CA, USA
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Estuardo Aguilar-Cordova;
Estuardo Aguilar-Cordova
2Advantagene, Inc, San Diego, CA, USA
3Harvard Gene Therapy Initiative, Harvard Medical School, Cambridge, MA, USA
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Perry B. Hackett;
Perry B. Hackett
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
4Discovery Genomics, Inc., Minneapolis, MN, USA
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R. Scott McIvor
R. Scott McIvor
1Gene Therapy Program and the Beckman Center for Transposon Research, Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall 321 Church Street S.E., Minneapolis, MN, 55455, USA
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Publisher: Portland Press Ltd
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 2005 Springer Science+Business Media, Inc.
2005
Biosci Rep (2004) 24 (6): 577–594.
Citation
Andrea D. Converse, Lalitha R. Belur, Jennifer L. Gori, Geyi Liu, Felipe Amaya, Estuardo Aguilar-Cordova, Perry B. Hackett, R. Scott McIvor; Counterselection and Co-Delivery of Transposon and Transposase Functions for Sleeping Beauty-Mediated Transposition in Cultured Mammalian Cells. Biosci Rep 1 December 2004; 24 (6): 577–594. doi: https://doi.org/10.1007/s10540-005-2793-9
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