Optimization of the Gal4/UAS transgenic tools in zebrafish

Zhang, Yunsheng; Ouyang, Jiawei; Qie, Jingrong; Zhang, Gongyuan; Liu, Liangguo; Yang, Pinhong

doi:10.1007/s00253-018-09591-0

Cited by 9 publications

(19 citation statements)

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“…However, the fact that high protein levels of Gal4 have certain toxicity toward cells coexpressing UAS-linked target genes and Gal4 protein cannot be ignored. Although some researchers have described and developed novel Gal4/UAS systems with smaller sizes but greater transactivation efficiency than the original system [18–20], little attention has been paid to the effects of Gal4 on the normal expression of nontarget genes. To objectively clarify the functions of target genes, it is necessary to determine whether and to what extent Gal4 protein expression affects normal nontarget gene expression.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

et al. 2020

Preprint

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25The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely 26 used to analyze gene function in many organisms, including the silkworm (Bombyx mori), a model 27 lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can 28 negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein 29 affects normal endogenous gene expression have rarely been studied. Here, we analyzed the 30 transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing 31 disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 32 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as 33 significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and 34 control groups, respectively. These DEGs were further annotated by gene function classification and 35 pathway assessment using public databases. In addition, 506 DEGs were shared (common) between 36 both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly 37 downregulated; many of them were annotated to be involved in metabolic processes such as "fat 38 digestion and absorption", "glycine, serine and threonine metabolism" and "glutathione metabolism" 39 and in signal transduction pathways such as the "Rap1 signaling pathway", "MAPK signaling 40 pathway" and "Hippo signaling pathway". Overall, this work enhances understanding of the effects 41 of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests 42 that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study 43 gene function. 44 Introduction 46The Gal4/upstream activating sequence(UAS) binary expression system, derived from yeast and 47 originally developed in Drosophila [1-3], is a powerful genetic tool that allows manipulation of 48 target gene expression in a spatiotemporally precise fashion. Since its first application in Drosophila, 49 the Gal4/UAS system has been widely used to analyze gene function in dozens of organisms, 50 including mice [4], zebrafish [5], Xenopus [6], Bombyx mori [7], Arabidopsis thaliana [8], 51 Triboliumcastaneum [9], Aedes aegypti [10], Anopheles stephensi [11] and Caenorhabditis elegans 52[12]. The Gal4/UAS system has also been employed to develop novel genetic tools, such as the 53 enhancer/gene trap system and the Q system [13][14][15], and it has been combined with genome editing 54 tools for conditional manipulation of gene expression in vivo [16][17]. 55 In recent decades, remarkable progress in gene function analysis has been achieved with the 56 Gal4/UAS system. However, the fact that high protein levels of Gal4 have certain toxicity toward 57 cells coexpressing UAS-linked target genes and Gal4 protein cannot be ignored. Although some 58 researchers have described and developed novel Gal4/UAS systems with smaller sizes but greater 59 transactivati...

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Section: Introductionmentioning

confidence: 99%

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

et al. 2020

Preprint

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“…The Gal4/UAS system has been employed in a diverse range of organisms, such as plants, drosophila, silkworm, xenopus, zebrafish and mice (Langenau, 2016;Fischer 4 et al, 1988;Ornitz, Moreadith and Leder, 1991;Scheer and Campos-Ortega, 1999;Hartley, Nutt and Amaya, 2002;Johnson et al, 2005;Ovchinnikov et al, 2008;Tsubota et al, 2014;Banerjee et al, 2017;Zhang et al, 2019). The development of the Tol2 transposon system as a transgenesis method revolutionized the use of the Gal4 system in these different animal models.…”

Section: Spatial Control With the Gal4/uas Systemmentioning

confidence: 99%

“…The Tol2 transposase was identified in the genome of the Japanese medaka fish and its discovery represented a breakthrough in the generation of transgenic lines (Koga et al, 1996;Kawakami et al, 2016). The high frequency of Tol2 transposon germline integration simplified the ability of isolating transgenics, which facilitated the development of Gal4 and UAS lines (Langenau, 2016;Kawakami et al, 2004;Zhang et al, 2019).…”

Section: Spatial Control With the Gal4/uas Systemmentioning

confidence: 99%

Section: Spatial Control With the Gal4/uas Systemmentioning

confidence: 99%

“…To further improve the system by increasing Gal4 activity, the Gal4 DNA-binding domain was fused to the transcriptional activation domain from the VP16 protein of the herpes simplex virus (Langenau, 2016;Zhang et al, 2019). In addition, the UAS promoter is commonly designed as repeats to increase expression efficiency of the gene of interest (Langenau, 2016;Zhang et al, 2019). Usually, 4x, 5x and 14x UAS repeats are used in order to achieve effective gene induction (Köster and Fraser, 2001;Asakawa et al, 2008;Daba, Koromilas and Pantopoulos, 2012;Zhang et al, 2019).…”

Section: Spatial Control With the Gal4/uas Systemmentioning

confidence: 99%

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CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3′-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5′-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.

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Optimization of the Gal4/UAS transgenic tools in zebrafish

Cited by 9 publications

References 38 publications

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

Beyond knockouts: Applying precision genome editing for conditional mutagenesis innovation in zebrafish

Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish

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