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A simple laboratory practical to illustrate RNA mediated gene interference using drosophila cell culture
Author(s) -
Buluwela Laki,
Kamalati Tahereh,
Photiou Andy,
Heathcote Dean A.,
Jones Michael D.,
Ali Simak
Publication year - 2010
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.20437
Subject(s) - rna interference , biology , gene , rna silencing , computational biology , rna , genetics , microbiology and biotechnology , schneider 2 cells , drosophila melanogaster
RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode in vitro transcription promoters fused to sequences from Drosophila genes of the Rho/Rac GTPase family. These DNA templates are then used to synthesize double stranded RNAs (dsRNA), which are subsequently used to transfect Drosophila Kc embryonic cells. The resulting RNAi produces simple cellular phenotypes that are observed following fluorescent histochemical staining. These phenotypes are ultimately related to gene ontology data that the students generate through a bioinformatic analysis of the sequences transcribed into dsRNA. Taken together, this laboratory exercise provides “hands on” experience of RNAi in a class setting and provides a framework for the in‐depth discussion of how this technique can be applied to studies of gene function. Biochemistry and Molecular Biology Education Vol. 38, No. 6, pp. 393‐399, 2010