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. 2019 Feb 5;9(3):e3158.
doi: 10.21769/BioProtoc.3158.

pNP Transgenic RNAi System Manual in Drosophila

Fang Wang  1 , Huan-Huan Qiao  1 , Rong-Gang Xu  1 , Jin Sun  1 , Ruibao Zhu  1 , Decai Mao  1 , Jian-Quan Ni  1   2
Affiliations

pNP Transgenic RNAi System Manual in Drosophila

Fang Wang et al. Bio Protoc. .

Abstract

Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.

Keywords: Drosophila; Gal4/UAS system; Multiple genes knockdown; Transgenic RNAi; pNP vector.

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Conflict of interest statement

Competing interestsThe authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. The map of pNP construct.
Vermilion is a selectable marker. attB sequence is used for phiC31 targeted integration at genomic attP landing sites. MCS1 allows a single shRNA to be cloned in both orientations, while with the help of MCS2 and the linker it could simultaneously generate multiple shRNAs once.
Figure 2.
Figure 2.. Parameters for short hairpins design
Figure 3.
Figure 3.. Overview of the selection procedures for generating Chromosome II transgenic flies
Figure 4.
Figure 4.. Overview of the selection procedures for generating Chromosome III transgenic flies
See this image and copyright information in PMC

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