As case studies, we show the assembly of inverted repeat constructs for profilin PRF1, actin ACT8 and ACT3, and actin-related protein ARP4 genes from Arabidopsis. IR-PCR allows assembly and cloning of RNAi constructs in any vector in a single day, as outlined in Fig.
We have devised a novel alternative strategy for making RNAi gene constructs, called inverted repeat PCR (IR-PCR), which requires only two rounds of PCR and one cloning step. These vectors require PCR of the target gene to introduce novel end sequences and multiple restriction enzyme-based or recombination-based DNA cloning steps that produce inefficiencies in assembling some sequences as inverted repeats ( Wesley et al. The demand of genomics for high-throughput analysis of gene function by RNAi has led to the development of several RNAi cloning vectors that express RNAs containing inverted repeats with stems derived from the target gene of interest. Furthermore, dissecting gene function using RNAi mediated gene silencing is often more efficient than isolating T-DNA insertion mutations of a target gene. In plants, the expression of stem-loop RNAs in which the double stranded RNA stem is homologous to the target gene or its RNA is more efficient at producing knockdown phenotypes than is antisense RNA ( Chuang and Meyerowitz 2000, Mette et al. RNAi can target specific RNA sequences for degradation or translational inhibition by a process called ‘post-transcriptional gene silencing’ (PTGS), or it can block gene expression in a related process termed ‘transcriptional gene silencing’ that affects promoter activity ( Meister and Tuschl 2004). RNA interference (RNAi) is now commonly used to knock down gene expression in plants, animals, and fungi ( Baulcombe 2004, Meister and Tuschl 2004).
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