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

doi:10.12300/j.issn.1674-5817.2025.116

Abstract

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

CLC Number:

Q964

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.

Figures/Tables 4

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.

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.

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.

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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