AI Article Synopsis
- Researchers are focusing on multiple gene silencing to effectively target metabolic pathways in eukaryotic organisms, necessitating the creation of refined RNA interference (RNAi) cassettes.
- A new two-step cloning strategy was developed to create multisite small interfering RNA (siRNA) cassettes, which optimizes the arrangement of siRNA sites in target messenger RNAs (mRNAs) for efficiency.
- This method utilizes one-step PCR with specially designed long primers, providing advantages in rapid screening, cost-effectiveness, and reduced procedural length, while also ensuring high stability and optimal structural validity of constructs in plants.
Article Abstract
Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning steps. In this method, effective siRNA sites in the target messenger RNAs (mRNAs) were determined using analysis and consecutively arranged to reduce length of inverted repeats. Here, we used one-step (polymerase chain reaction) PCR by designed long primer sets covering the selected siRNA sites. Rapid screening, cost-effective and shorten procedure are advantages of this method compare to PCR classic cloning. Validity of constructs was confirmed by optimal centroid secondary structures with high stability in plants.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019331 | PMC |
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