Gal4/UAS Transgenic Tools and Their Application to Zebrafish

Halpern, Marnie E.; Rhee, Jerry M.; Goll, Mary; Akitake, Courtney M.; Parsons, Michael; Leach, Steven D.

doi:10.1089/zeb.2008.0530

Cited by 179 publications

(145 citation statements)

References 127 publications

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“…Bipartite gene expression systems allow selective gene expression in a tissue-specific manner in vivo (reviewed by del Valle Rodríguez et al, 2012;Elliott, 2008;Halpern et al, 2008). They consist of two parts: a driver line and an effector/reporter line.…”

Section: Introductionmentioning

confidence: 99%

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

et al. 2014

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The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.

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Section: Introductionmentioning

confidence: 99%

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

et al. 2014

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“…such as Xenopus and zebrafish (63,64), to study signaling and development. By expressing GAL4 from a tissue-specific driver, spatial resolution of expression of the UAS target gene can be achieved; however, this system lacks temporal resolution.…”

Section: Discussionmentioning

confidence: 99%

Rational elicitation of cold-sensitive phenotypes

Baliga

Majhi

Mondal

et al. 2016

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

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Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.conditional mutants | rational design | cold sensitivity | heat-induced expression | transfer between organisms C onditional mutants are powerful tools for studying gene function in vivo. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and lacks that function under a different set of conditions, called restrictive, whereas the wild type (WT) phenotype is similar across both conditions. Cold-sensitive (cs) mutants behave like loss-of-function mutants at temperatures lower than a cutoff temperature, but have WT-like phenotypes at higher temperatures. In contrast, temperature-sensitive (ts) mutants show heat sensitivity and behave like the WT below the restrictive temperature. Both cs and ts mutants can provide important information about protein structure, function, and assembly. cs mutants are rarer than ts mutants, and the molecular basis for generation of cs phenotypes is currently unclear.cs mutants have been used to analyze various biological phenomena, most commonly the cell cycle (1-3), ribosome assembly (4-8), and protein export (9-11), as well as to understand macromolecular structure-function relationships (12, 13). Various ts and cs variants also have helped us understand P22 phage coat protein assembly. Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20). Cold sensitivity also has been attribut...

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“…The Gal4/UAS system, a binary system providing temporal and spatial manipulation of gene expression, has been extensively employed in zebrafish (Halpern et al 2008;Baier and Scott 2009). An optimized Gal4/UAS system, the KalTA4/ 4xUAS system, was developed to balance transcriptional activity with toxicity (Distel et al 2009).…”

Section: Discussionmentioning

confidence: 99%

“…The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of genes in Drosophila (Duffy 2002), mouse (Lewandoski 2001) and zebrafish (Halpern et al 2008;Baier and Scott 2009). In general, the Gal4/UAS technique is based on two transgenic strains: an activator strain expressing Gal4 under the control of a temporally and spatially regulated promoter or enhancer and an effector strain carrying a gene of interest regulated by a repeated UAS and a mini-promoter.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish

et al. 2015

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The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes. In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences. We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations. In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains. We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences. Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm. Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization. Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UASspecific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish.

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Gal4/UAS Transgenic Tools and Their Application to Zebrafish

Cited by 179 publications

References 127 publications

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

Rational elicitation of cold-sensitive phenotypes

Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish

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