UAS-Irk3-EGFP转基因果蝇品系的构建与鉴定

doi:10.12300/j.issn.1674-5817.2025.116

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (6): 656-662.DOI: 10.12300/j.issn.1674-5817.2025.116

• 无脊椎实验动物：果蝇 • 上一篇下一篇

UAS-Irk3-EGFP转基因果蝇品系的构建与鉴定

王明珠¹^,²(), 高英豪¹(), 谭霜霜¹, 吴薇¹()

^1.中国科学院分子细胞科学卓越创新中心/生物化学与细胞生物学研究所, 上海 200031
^2.中国科学院大学, 北京 100049

收稿日期:2025-07-14 修回日期:2025-10-17 出版日期:2025-12-25 发布日期:2025-12-19
通讯作者: 吴薇（1980—），女，博士，高级工程师，研究方向为果蝇基因编辑、果蝇资源库管理和果蝇发育生物学。E-mail：wuwei@sibcb.ac.cn。ORCID：0009-0000-6761-3993
作者简介:王明珠（1993—），女，硕士，研究方向为果蝇发育生物学。E-mail：wangmingzhu2022@sibcb.ac.cn。ORCID：0009-0003-4987-0605；
高英豪（1996—），男，硕士，助理工程师，研究方向为果蝇发育生物学。E-mail：gaoyinghao@sibcb.ac.cn。ORCID：0009-0006-1941-5283
基金资助:
国家重点研发计划“果蝇和线虫发育代谢资源库的系统构建与分析”(2021YFA0805800)

Construction and Characterization of UAS-Irk3-EGFP Transgenic Drosophila Lines

WANG Mingzhu¹^,²(), GAO Yinghao¹(), TAN Shuangshuang¹, WU Wei¹()

^1.Center for Excellence in Molecular Cell Science/Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Shanghai 200031, China
^2.University of Chinese Academy of Sciences, Beijing 100049, China

Received:2025-07-14 Revised:2025-10-17 Published:2025-12-25 Online:2025-12-19
Contact: WU Wei (ORCID: 0009-0000-6761-3993), E-mail: wuwei@sibcb.ac.cn

摘要/Abstract

摘要：

目的建立UAS-Irk3-EGFP转基因果蝇品系，为转基因果蝇品系的构建与鉴定提供标准化流程，并为内向整流钾通道（inwardly rectifying potassium channel，Irk）3基因的功能研究提供果蝇模型支撑，丰富国家果蝇资源。方法利用PCR技术，从果蝇cDNA文库中扩增出Irk3基因的编码序列（coding sequences，CDS）。利用同源重组技术将其与增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因一起克隆到pUAST-attB载体中。通过显微注射技术，将获得的重组质粒注射入果蝇品系attP-25C6的胚胎中，利用红眼表型筛选能表达UAS-Irk3-EGFP的转基因红眼果蝇，然后通过与平衡子果蝇杂交进行背景纯化和平衡保种，最后通过PCR扩增和翅成虫盘免疫荧光染色方法鉴定其正确性。结果构建了pUAST-attB-Irk3-EGFP重组质粒，并成功获得UAS-Irk3-EGFP转基因果蝇品系。PCR扩增结果证实，Irk3-EGFP基因序列已成功整合到转基因果蝇品系的基因组中。翅成虫盘免疫染色实验发现，翅囊区特异性表达的MS1096-GAL4驱动Irk3基因在翅成虫盘翅囊表达，后隔间特异性表达的hh-GAL4可驱动Irk3基因在翅成虫盘后隔间表达。结论成功构建了UAS-Irk3-EGFP转基因果蝇品系，为应用半乳糖凝集素4（galectin-4，GAL4）/上游激活序列（upstream activating sequence，UAS）基因表达调控系统深入研究Irk3基因功能奠定了基础。

关键词: 黑腹果蝇, 内向整流钾通道3, UAS-Irk3-EGFP转基因果蝇, GAL4/UAS系统

Abstract:

Objective To establish a UAS-Irk3-EGFP transgenic Drosophila line, provide a standardised procedure for the construction and characterization of transgenic Drosophila lines, support functional studies of the inwardly rectifying potassium channel (Irk) 3 gene in Drosophila, and enrich national Drosophila resources. Methods Using PCR technology, coding sequence (CDS) of Irk3 gene was amplified from Drosophila cDNA library. The sequence was then cloned together with the enhanced green fluorescent protein (EGFP ) gene into pUAST-attB vector via homologous recombination. By microinjection, the recombinant plasmid was injected into embryos of attP-25C6 Drosophila line. Transgenic red-eyed flies expressing UAS-Irk3-EGFP were screened by red eye phenotype, followed by background purification and balanced preservation by crossing with balancer flies. Finally, correctness was verified by PCR amplification and immunofluorescence staining of wing imaginal discs. Results The pUAST-attB-Irk3-EGFP recombinant plasmid was constructed, and the UAS-Irk3-EGFP transgenic Drosophila line was successfully obtained. PCR amplification results confirmed that the Irk 3-EGFP gene sequence was successfully integrated into the genome of the transgenic flies. Immunostaining experiment of wing imaginal discs showed that MS1096-GAL4, which was specifically expressed in the wing pouch, could drive Irk3 gene expression in the wing pouch of the wing imaginal disc, and hh-GAL4, which was specifically expressed in the posterior compartment, could drive target gene expression in the posterior compartment of the wing imaginal disc. Conclusion The UAS-Irk3-EGFP transgenic Drosophila line is successfully established, laying a foundation for in-depth studies of Irk3 gene function using galectin-4 (GAL4)/upstream activating sequence （UAS) gene expression regulation system.

Key words: Drosophila melanogaster, Inwardly rectifying potassium channel 3, UAS-Irk3-EGFP transgenic Drosophila, GAL4/UAS system

中图分类号:

Q964

王明珠,高英豪,谭霜霜,等. UAS-Irk3-EGFP转基因果蝇品系的构建与鉴定[J]. 实验动物与比较医学, 2025, 45(6): 656-662. DOI: 10.12300/j.issn.1674-5817.2025.116.

WANG Mingzhu,GAO Yinghao,TAN Shuangshuang,et al. Construction and Characterization of UAS-Irk3-EGFP Transgenic Drosophila Lines[J]. Laboratory Animal and Comparative Medicine, 2025, 45(6): 656-662. DOI: 10.12300/j.issn.1674-5817.2025.116.

图/表 4

图1 Irk通道的亚单位结构示意图注：TM1和TM2均为跨膜区结构域，H5为胞外孔形成区域H5环。

Figure 1 Schematic diagram of the subunit structure of the inwardly rectifying potassium channel channelNote： TM1 and TM2 are both transmembrane domains， and H5 is the H5 loop of the extracellular pore-forming region.

图2 基因扩增与线性化载体的琼脂糖凝胶电泳结果注：A，EGFP基因扩增产物；B，Irk3基因扩增产物；C，线性化载体pUAST-attB；D，pUAST-attB-Irk3-EGFP质粒构建模式图。

Figure 2 Agarose gel electrophoresis results of gene amplification and linearized vectorNote： A， Amplified product of EGFP gene； B， Amplified product of Irk3 gene； C， Linearized vector pUAST-attB； D， Schematic diagram of pUAST-attB-Irk3-EGFP plasmid construction.

图3 UAS-Irk3-EGFP转基因果蝇品系的阳性筛选及PCR鉴定注：A，attP-25C6背景的果蝇；B，含有UAS-Irk3-EGFP的红眼果蝇；C，UAS-Irk3-EGFP转基因果蝇品系的PCR鉴定电泳图。标尺大小为100 μm。

Figure 3 Positive screening and PCR identification of the UAS-Irk3-EGFP transgenic Drosophila lineNote： A， Drosophila with attP-25C6 background； B， Red-eyed Drosophila with UAS-Irk3-EGFP；C， PCR identification electrophoresis pattern of UAS-Irk3-EGFP transgenic Drosophila line. Scale bar：100 μm.

图4 转基因果蝇的荧光验证注：A，MS1096-GAL4驱动Irk3-EGFP在翅囊区表达；B，hh-GAL4驱动Irk3-EGFP在后隔间表达；C，MS1096-GAL4驱动子特异性表达的区域模式图；D，hh-GAL4驱动子特异性表达的区域模式图。绿色区域为Irk3-EGFP蛋白的荧光信号，蓝色区域为细胞核的免疫染色信号（DAPI）。标尺大小为50 μm。

Figure 4 Fluorescent verification of transgenic DrosophilaNote： A， MS1096-GAL4 drives the expression of Irk3-EGFP in the wing pouch region； B， hh-GAL4 drives Irk3-EGFP expression in the posterior compartment； C， Regional pattern diagram of MS1096-GAL4 driver-specific expression； D， Regional pattern diagram of hh-GAL4 driver-specific expression. The green areas indicate fluorescent signal of Irk3-EGFP protein， and the blue areas indicate immuno-staining signal of cell nucleus （DAPI）. Scale bar： 50 μm.

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UAS-Irk3-EGFP转基因果蝇品系的构建与鉴定

Construction and Characterization of UAS-Irk3-EGFP Transgenic Drosophila Lines

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