Toward a Functional Annotation of the Human Genome Using Artificial Transcription Factors

Lee, Dong-Ki; Park, Jin Woo; Kim, Youn‐Jae; Kim, Jiwon; Lee, Yangsoon; Kim, Jeonglim

doi:10.1101/gr.1397903

Cited by 15 publications

(9 citation statements)

References 35 publications

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“…Of these 54 ZF modules, 31 were derived from DNA sequences in the human genome, and two were from those in the Drosophila genome (termed ''ToolGen modules''). These naturally occurring ZFs have been used repeatedly for the regulation of many genes in bacterial, yeast, and mammalian cells (Bae et al 2003;Lee et al 2003Lee et al , 2004Park et al 2003Park et al , 2005aKwon et al 2006;Yun et al 2008). The remaining 21 ZF modules were either selected from libraries of ZF variants using phage display (Barbas modules) (Dreier et al 2000(Dreier et al , 2001(Dreier et al , 2005Liu et al 2002) or produced by site-directed mutagenesis (Sangamo modules) , and were chosen to supplement the DNA-binding specificities of ToolGen modules.…”

Section: Zfn Design and Synthesismentioning

confidence: 99%

Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly

Kim

Lee²,

Kim³

et al. 2009

Genome Res.

418

328

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Broad applications of zinc finger nuclease (ZFN) technology-which allows targeted genome editing-in research, medicine, and biotechnology are hampered by the lack of a convenient, rapid, and publicly available method for the synthesis of functional ZFNs. Here we describe an efficient and easy-to-practice modular-assembly method using publicly available zinc fingers to make ZFNs that can modify the DNA sequences of predetermined genomic sites in human cells. We synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene-a co-receptor required for HIV infection-and found that many of these nucleases induced site-specific mutations in the CCR5 sequence.

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Section: Zfn Design and Synthesismentioning

confidence: 99%

Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly

Kim

Lee²,

Kim³

et al. 2009

Genome Res.

418

328

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show abstract

“…Recent advances in this field now allow one to construct randomized ZFP-transcription factor (ZFP-TFs) libraries that consist of tens of thousands of distinct transcription factors (31)(32)(33). These libraries have been used successfully to screen for components that induce diverse phenotypes of interest, such as cellular differentiation, stress resistance (31), over-expression of surface antigens (32), or resistance to Taxol, an anti-cancer agent (33). The selected ZFP-TFs could bind to and modulate expression of their target genes, which would generate phenotypes of interest.…”

Section: Introductionmentioning

confidence: 99%

Identification and Use of Zinc Finger Transcription Factors That Increase Production of Recombinant Proteins in Yeast and Mammalian Cells

Park

Seol

Yang

et al. 2008

Biotechnol Progress

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Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells.

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“…showed that regulation of a single target gene within the entire human genome is achievable using a 6-finger ZFP transcriptional repressor that recognize an 18-bp site in the genome (32). The ability of ZFPs to activate or repress target gene expression was also exploited in a recent report, where the method was used to identify novel functional relationships among genes (33). …”

Section: Discussionmentioning

confidence: 99%

Suppression of vascular endothelial growth factor expression at the transcriptional and post-transcriptional levels

Kwon

Shin

Kim

2005

Nucleic Acids Research

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Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.

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Toward a Functional Annotation of the Human Genome Using Artificial Transcription Factors

Cited by 15 publications

References 35 publications

Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly

Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly

Identification and Use of Zinc Finger Transcription Factors That Increase Production of Recombinant Proteins in Yeast and Mammalian Cells

Suppression of vascular endothelial growth factor expression at the transcriptional and post-transcriptional levels

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