Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.
We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. jbai@genetics.med.harvard.edu
Jianwu
J
Bai
Katharine J
KJ
Sepp
Norbert
N
Perrimon
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation; D002478:Cells, Cultured; D003470:Culture Media; D004330:Drosophila / Q000166:cytology / Q000196:embryology*; D005775:Gastrula / Q000166:cytology*; D023281:Genomics; D034622:RNA Interference; D012330:RNA, Double-Stranded
D003470:Culture Media; D012330:RNA, Double-Stranded