split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.
split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.
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DOI：
10.1073/pnas.2304730120
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发表时间：
2023-06-13
影响因子：
11.1
通讯作者：
Perrimon, Norbert
中科院分区：
文献类型：
--
作者：
Ewen-Campen, Ben;Luan, Haojiang;Xua, Jun;Singh, Rohit;Joshi, Neha;Thakkar, Tanuj;Berger, Bonnie;White, Benjamin H.;Perrimon, Norbert
split-Gal4 系统使果蝇研究人员能够以非凡的细胞类型特异性驱动转基因表达。然而，现有的 split-Gal4 系统无法进行时间控制，因此无法应用于许多重要的研究领域。在这里，我们提出了一种基于自切除分裂内含肽的 split-Gal4 系统，该系统可由 Gal80 控制，以及相关的药物诱导分裂 GeneSwitch 系统。这种方法既可以利用单细胞 RNAseq 数据集，也可以为单细胞 RNAseq 数据集提供信息，并且我们引入了一种算法来识别精确且狭窄地标记所需细胞簇的基因对。我们的分裂内含肽 Gal4 系统将对果蝇研究界有价值，并允许创建高度特异性的遗传驱动因素，这些驱动因素也是可诱导/可抑制的。 split-Gal4 系统允许对果蝇中高度特异性的细胞类型和组织进行交叉遗传标记。然而，现有的分裂Gal4系统与标准Gal4系统不同，不能被Gal80抑制，因此不能被暂时控制。这种时间控制的缺乏妨碍了 split-Gal4 实验，在这种实验中，基因操作必须限制在特定的时间点。在这里，我们描述了一种基于自切除分裂内含肽的 split-Gal4 系统，该系统与当前 split-Gal4 系统和 Gal4 试剂一样强烈地驱动转基因表达，但可被 Gal80 抑制。我们使用荧光报告基因并通过肠道中的可逆肿瘤诱导来证明“分裂内含肽 Gal4”在体内的有效诱导能力。此外，我们表明我们的分裂内含肽 Gal4 可以扩展到药物诱导的 GeneSwitch 系统，为具有诱导控制的交叉标记提供了一种独立的方法。我们还表明，分裂内含肽 Gal4 系统可用于基于单细胞 RNAseq (scRNAseq) 数据集生成的计算机预测生成高度细胞类型特异性的遗传驱动因素，并且我们描述了一种算法（“两个背景”或 TAB）来预测跨多个组织特异性 scRNA 数据集的簇特异性基因对。我们提供了一个质粒工具包，可基于目标基因的 CRISPR 敲入或使用增强子片段来高效创建分裂内含肽 Gal4 驱动程序。总而言之，分裂内含肽 Gal4 系统允许创建可诱导/可抑制的高度特异性交叉遗传驱动因素。
The split-Gal4 system allows Drosophila researchers to drive transgene expression with extraordinary cell type specificity. However, the existing split-Gal4 system cannot be controlled temporally, and therefore cannot be applied to many important areas of research. Here, we present a split-Gal4 system based on a self-excising split-intein, which is controllable by Gal80, as well as a related drug-inducible split GeneSwitch system. This approach can both leverage and inform single-cell RNAseq datasets, and we introduce an algorithm to identify pairs of genes that precisely and narrowly mark a desired cell cluster. Our split-intein Gal4 system will be of value to the Drosophila research community, and allow for the creation of highly specific genetic drivers that are also inducible/repressible. The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of “split-intein Gal4” in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type–specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm (“Two Against Background” or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.
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影响因子：
3.3
作者：
Dionne H;Hibbard KL;Cavallaro A;Kao JC;Rubin GM
通讯作者：
Rubin GM
影响因子：
8.8
作者：
Diao F;Ironfield H;Luan H;Diao F;Shropshire WC;Ewer J;Marr E;Potter CJ;Landgraf M;White BH
通讯作者：
White BH
DOI：
10.1093/g3journal/jkac102
发表时间：
2022-05-30
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
通讯作者：
--
影响因子：
5.6
作者：
CAREY, M;KAKIDANI, H;PTASHNE, M
通讯作者：
PTASHNE, M
影响因子：
7
作者：
Aevermann B;Zhang Y;Novotny M;Keshk M;Bakken T;Miller J;Hodge R;Lelieveldt B;Lein E;Scheuermann RH
通讯作者：
Scheuermann RH