ArticleMolecular and biochemical parasitology2012
A single-cloning-step procedure for the generation of RNAi plasmids producing long stem-loop RNA.
Article in Molecular and biochemical parasitology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 10 citations in OpenAlex.
- A long noncoding RNA promotes parasite differentiation in African trypanosomes.Science advances · 2022Article
- Identification of positive and negative regulators in the stepwise developmental progression towards infectivity in Trypanosoma brucei.Scientific reports · 2021Article
- Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis.Memorias do Instituto Oswaldo Cruz · 2019Article
- Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.PLoS pathogens · 2015Article
- Article
- A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly.Molecular and biochemical parasitologyArticle
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
RNA interference (RNAi), used as a tool, has revolutionized the studies of gene function. Long stem-loop dsRNA has been proven the most effective trigger for down-regulating target transcripts in RNAi-positive trypanosomatid parasites. Here we describe a protocol for constructing plasmids that produce long stem-loops by using a single cloning step. Inverted repeats are first obtained by self-ligation of PCR products that contain a randomized segment at one of their ends and then inserted in a plasmid vector. The random sequences create the loop (or "stuffer") of the hairpin. This methodology was tested in Leishmania (Viannia) braziliensis to constitutively knock down the mRNAs for the well-studied paraflagellar rod protein 1 and 2 (PFR1 and PFR2) genes and revealed that mRNA cleavage products are unusually stable in these parasites. The protocol is suitable for any plasmid (for constitutive or inducible expression) and for any organism in which long stem-loops can be used to elicit RNAi.
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Registered trials
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