Synthetic design of strong promoters
- 21 January 2010
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 107 (6), 2538-2543
- https://doi.org/10.1073/pnas.0914803107
Abstract
We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-mer DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types.Keywords
This publication has 22 references indexed in Scilit:
- Targeted manipulation of mammalian genomes using designed zinc finger nucleasesBiochemical and Biophysical Research Communications, 2009
- Diversity-based, model-guided construction of synthetic gene networks with predicted functionsNature Biotechnology, 2009
- Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factorsNature Protocols, 2009
- Analysis of combinatorial cis-regulation in synthetic and genomic promotersNature, 2008
- Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleasesNucleic Acids Research, 2006
- Second-generation shRNA libraries covering the mouse and human genomesNature Genetics, 2005
- Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarraysNature Genetics, 2004
- Analysis of Different Promoter Systems for Efficient Transgene Expression in Mouse Embryonic Stem Cell LinesThe International Journal of Cell Cloning, 2002
- Lentiviral Vectors for Enhanced Gene Expression in Human Hematopoietic CellsMolecular Therapy, 2000
- GAL4-VP16 is an unusually potent transcriptional activatorNature, 1988