Gene silencing for functional analysis: assessing RNAi as a tool for manipulation of gene expression.
The availability of a large number of gene-disrupted mutants (either from natural mutants' collections or from knockout projects) is a great advantage for functional analysis studies. However, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. Therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose impaired expression confers a pleiotropic phenotype, is extremely beneficial for studying their function. Here, we describe the use of RNAi as a gene silencing mechanism, in a manner that might facilitate the functional analysis of such essential genes. Two alternative strategies for the construction of an RNAi-induced inverted-repeat construct are demonstrated and a third alternative is suggested. In addition, DNA-mediated transformation of conidia by electroporation, RNA extraction from fungal mycelium and northern blot analysis are described in detail.The experimental results presented, demonstrate that RNAi can be employed as a gene silencing tool in Neurospora crassa, both for nonessential (al-2) and essential (cot-1) genes, resulting in a range of stable, partially silenced mutants, exhibiting different phenotypes.
Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Carmit
C
Ziv
Oded
O
Yarden
D015152:Blotting, Northern; D015870:Gene Expression; D020868:Gene Silencing; D005800:Genes, Fungal; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D009492:Neurospora crassa / Q000235:genetics / Q000254:growth & development; D010641:Phenotype; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012091:Repetitive Sequences, Nucleic Acid; D014170:Transformation, Genetic
D050505:Mutant Proteins; D034741:RNA, Small Interfering