New tool for an old problem: can RNAi efficiently resolve the issue of genetic redundancy?

Kan, Lixin; Kessler, John A.

doi:10.1002/bies.20172

Cited by 5 publications

(2 citation statements)

References 30 publications

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“…A particularly useful application is to elucidate the functions of gene paralog families. As long as the family members are sufficiently related, a single siRNA can simultaneously knockdown all family members and thereby unveil the redundant function of the gene paralog family [ 1 – 3 ].…”

Section: Introductionmentioning

confidence: 99%

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

et al. 2015

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RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.

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Section: Introductionmentioning

confidence: 99%

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

et al. 2015

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“…Several members of the same gene family or completely different genes may code different proteins, which can perform similar functions in the organism. This redundancy could result in compensation of the loss of function of the targeted genes, leading to a masking of the silenced phenotype (Kan & Kessler, 2005). The influence of the presence of redundant proteins was observed in the knockdown of salivary apyrase of the mosquito A. gambiae (Boisson et al , 2006).…”

Section: Methods For Achieving Rnai In Triatominesmentioning

confidence: 99%

Application of RNA interference in triatomine (Hemiptera: Reduviidae) studies

et al. 2012

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Triatomines (Hemiptera: Reduviidae) are obligate hematophagous insects. They are of medical importance because they are vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. In recent years, the RNA interference (RNAi) technology has emerged as a practical and useful alternative means of studying gene function in insects, including triatomine bugs. RNAi research in triatomines is still in its early stages, several issues still need to be elucidated, including the description of the molecules involved in the RNAi machinery and aspects related to phenotype evaluation and persistence of the knockdown in different tissues and organs. This review considers recent applications of RNAi to triatomine research, describing the major methods that have been applied during the knockdown process such as the double-stranded RNA delivery mechanism (injection, microinjection, or ingestion) and the phenotype characterization (mRNA and target protein levels) in studies conducted with the intent to provide greater insights into the biology of these insects. In addition to the characterization of insect biomolecules, some with biopharmacological potential, RNAi may provide a new view of the interaction between triatomine and trypanosomatids, enabling the development of new measures for vector control and transmission of the parasite.

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Analysis of Mouse Development with Conditional Mutagenesis

Lewandoski

Conditional Mutagenesis: An Approach to Disease Models

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Explorations into the molecular embryology of the mouse have played a vital role in our understanding of the basic mechanisms of gene regulation that govern development and disease. In the last 15 years, these mechanisms have been analyzed with vastly greater precision and clarity with the advent of systems that allow the conditional control of gene expression. Typically, this control is achieved by silencing or activating the gene of interest with site-specific DNA recombination or transcriptional transactivation. In this review, I discuss the application of these technologies to mouse development, focusing on recent innovations and experimental designs that specifically aid the study of the mouse embryo.

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New tool for an old problem: can RNAi efficiently resolve the issue of genetic redundancy?

Cited by 5 publications

References 30 publications

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

Application of RNA interference in triatomine (Hemiptera: Reduviidae) studies

Analysis of Mouse Development with Conditional Mutagenesis

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