期刊
G3-GENES GENOMES GENETICS
卷 6, 期 8, 页码 2467-2478出版社
GENETICS SOCIETY AMERICA
DOI: 10.1534/g3.116.028571
关键词
CRISPR; stem cell; Drosophila; loss-of-function
资金
- Human Frontier Science Program long-term fellowship
- Boehringer Ingelheim Fonds PhD fellowship
- Austrian Academy of Sciences
- Austrian Science Fund [I_1281-B19, Z_153_B09]
- advanced grant from the European Research Council
- Austrian Science Fund (FWF) [Z153] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [Z 153] Funding Source: researchfish
Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.
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