期刊
CYTOMETRY PART A
卷 87A, 期 5, 页码 405-418出版社
WILEY-BLACKWELL
DOI: 10.1002/cyto.a.22650
关键词
episome; expression kinetics; integration-deficient; multicolor flow cytometry; proliferation rate; protein half-life; retroviral vector; transient gene expression
资金
- German Academic Exchange Service (DAAD, Modern Application of Biotechnology)
- Federal Ministry of Education and Research (BMBF)
- Deutsche Forschungsgemeinschaft [SFB738, SPP1230]
- Cluster of Excellence REBIRTH
- European Union
- Hannover Biomedical Research School (HBRS
- DFG) [GSC 108]
- PhD program Molecular Medicine
Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while avoiding integration, the developed technology is of great significance for a broad panel of applications, including transcription-factor-based induced cell fate conversion and controlled transfer of genetically encoded RNA- or protein-based drugs. (c) 2015 International Society for Advancement of Cytometry
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