Controlling gene expression in plants using synthetic zinc finger transcription factors

Stege, Justin T.; Guan, Xuen; Ho, Thao N. T.; Beachy, Roger N.; Barbas, Carlos F.

doi:10.1046/j.1365-313x.2002.01492.x

Cited by 64 publications

(41 citation statements)

References 39 publications

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“…Where necessary, increased specificity can be achieved by using four-finger ZFNs that recognize sequences of 24 bp. A number of different synthetic transcription factors with zinc-finger DNA-binding domains have been shown to function in plant cells (49)(50)(51)(52), supporting the idea that zinc fingers can provide access to many genomic targets.…”

Section: Dsb Repair In Plantsmentioning

confidence: 87%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

386

249

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Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFNinduced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.gene targeting ͉ nonhomologous end joining A major focus of plant biotechnology is genetic modification and improvement of crop plants. With this aim, large-scale genome and͞or EST sequencing projects are underway for many important plant species including rice, maize, wheat, soybean, and tomato (www.ncbi.nlm.nih.gov͞genomes͞PLANTS͞ PlantList.html). The enormous amount of genome-sequence information becoming available has intensified the need for methods that can use this sequence information to generate targeted modifications in plant genes. Targeted-mutagenesis methods could be used experimentally to investigate plant gene function or for genetic modification of important crop plants. Such methods are especially important for species, including most crops, that lack readily available mutant collections (1, 2). Furthermore, targeted mutagenesis could facilitate development of genetically modified crops lacking transgenic DNA, including genes conferring resistance to antibiotics. To date, efficient and widely usable methods for targeted modification of higher plant genomes are not available.The most widely used targeted-mutagenesis strategy is gene targeting (GT) by homologous recombination (3-6). Efficient GT procedures have been available for Ͼ20 years in yeast (7) and mouse (8). In these systems, DNA fragments are introduced into cultured cells, and repair by homologous recombination causes the introduced DNA to be incorporated into the homologous locus. Typically, GT events ...

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Section: Dsb Repair In Plantsmentioning

confidence: 87%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

386

249

View full text Add to dashboard Cite

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“…The higher transactivation observed for ATF-97 could be explained by its close proximity to the transcription start site, facilitating the recruitment of the polymerase II complex (Stege et al, 2002). To study if the designed ATFs were able to upregulate endogenous maspin in breast cancer cell lines, we expressed the ATFs using the retroviral vector pMX-IRES-GFP (Royer et al, 2004), which allows the tracking of transduced cells by flow cytometry.…”

Section: Atf -97mentioning

confidence: 99%

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

et al. 2006

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The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

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“…Appending a transcriptional activator, such as the VP16 transactivation domain (47), or a repressor, such as the Kruppel-associated box (KRAB) domain (38), allows potent up-or down-regulation of a gene of interest (3)(4)(5)34). Such artificial transcription factors have been shown to regulate numerous endogenous genes in many different animal and plant cells (3,22,23,28,53,61).…”

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confidence: 99%

Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

Eberhardy

Gonçalves

Coelho

et al. 2006

J Virol

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The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjacent to the PBS and repress transcription from the HIV-1 long terminal repeat (LTR). These proteins bound to the LTR in vivo, as demonstrated by the chromatin immunoprecipitation assay. In transient reporter assays, three of the four proteins repressed transcription of a reporter driven by the HIV-1 LTR. Only one of these proteins, however, designated KRAB-PBS2, was able to prevent virus production when transduced into primary lymphocytes. We observed >90% inhibition of viral replication over the course of several weeks compared to untransduced cells, and no significant cytotoxicity was observed. Long-term exposure of HIV-1 to KRAB-PBS2 induced mutations in the HIV-1 PBS that reduced the effectiveness of the repressor, but these mutations also resulted in decreased rates of viral replication. These results show that KRAB-PBS2 has the potential to be used in antiviral therapy for AIDS patients and might complement other gene-based strategies.

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Controlling gene expression in plants using synthetic zinc finger transcription factors

Cited by 64 publications

References 39 publications

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

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