Gene silencing for functional analysis: assessing RNAi as a tool for manipulation of gene expression.

摘要: The availability of a large number gene-disrupted mutants (either from natural mutants' collections or knockout projects) is great advantage for functional analysis studies. However, disfunction many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes cell morphology, conferring major difficulty studying null mutants. Therefore, obtaining variable levels reduction gene expression, especially essential genes whose impaired expression confers pleiotropic phenotype, extremely beneficial their function. Here, we describe the use RNAi as silencing mechanism, manner that might facilitate such genes. Two alternative strategies construction an RNAi-induced inverted-repeat construct are demonstrated third suggested. In addition, DNA-mediated transformation conidia by electroporation, RNA extraction mycelium northern blot described detail.The experimental results presented, demonstrate can be employed tool Neurospora crassa, both nonessential (al-2) (cot-1) resulting range stable, partially silenced mutants, exhibiting different phenotypes.