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The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
The Advent of RNAi In Entomology (Insects)
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The Advent of RNAi In Entomology (Insects)

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Good for Basic Knowledge of Rnai In insects

Good for Basic Knowledge of Rnai In insects

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  • They found that the injection of dsRNA corresponding to the vermilion eye color gene of first instar nymphs triggered a suppression of ommochrome
    formation in the eye lasting through two instars, equivalent
    to 10–14 days.
  • This study provided an important insight about how regenerating blastemal cells are aware of both their position and the normal size of the leg.
    Because the Ds/Ft system is conserved in vertebrates,
    their results provided clues to the mechanisms of regeneration,
    which are relevant to vertebrate systems (Bando
    et al. 2009).
  • Transcript

    • 1. The advent of RNA interference in Entomology Justus Liebig University, Giessen (Germany) By: Ravi Dhiman Harmeet Singh chawla
    • 2. Introduction: Outline  What is RNAi?  Brief History  Pathways of RNAi  RNAi -Beginning in Insects
    • 3. What Is RNAi?  RNA interference (RNAi) is a highly evolutionarily conserved mechanism of gene regulation  There are two main classes of small non-coding RNAs (sRNAs) found in insects namely microRNAs (miRNAs) and short interfering RNAs (siRNAs)  The biogenesis of miRNAs and siRNAs differs in the way they enter the RNAi pathway, both have common elements  miRNAs and siRNAs are generated from larger more complex dsRNA by the ribonuclease III enzyme Dicer  Both then associates with an Argonaut family protein (AGO) to become part of the RISC- which functions to down regulate gene expression
    • 4. Need for RNAi  Defense Mechanism – As a defense mechanism to protect against transposons and other insertional elements – Protects against viral infections  Genome Wide Regulation – RNAi plays a role in regulating development and genome maintenance
    • 5. Brief History of RNAi Source: Invitrogen
    • 6. siRNA Pathway miRNA Pathway Plants Animals Plants and Animals
    • 7. RNAi -Beginning in Insects  Andrew Fire and Craig Mello first described the RNAi phenomenon in the nematode worm C.elegans. Injected into C.elegans dsRNA led to an efficient sequence-specific silencing.  And coined the term "RNA Interference" RNAi can operate in insects too!
    • 8. RNAi was found to work in many diverse species Fungi Trypanosomes Zebrafish Mice
    • 9. Fire and Mello Experiment Source: nobleprize.org
    • 10. Andrew FireCraig Mello Happy Ending
    • 11. RNAi-mediated gene knockdown  RNAi has been readily utilised to investigate gene function in insects  Effective RNAi has been demonstrated in many insect orders  Diptera (Lum et al., 2003; Dietzl et al., 2007)  Lepidoptera (Chen et al., 2008; Yu et al., 2008; Tian et al., 2009; Terenius et al., 2011)  Coleoptera (Arakane et al., 2004; Suzuki et al., 2008)  Hymenoptera (Schluns & Crozier, 2007; Antonio et al., 2008)  But has also been performed in insects in a variety of developmental stages
    • 12. RNAi in Insects 12
    • 13. Parental RNAi From Shinmyo Y, Mito T, Matsushita T et al. (2005) caudal is required for gnathal and thoracic patterning and for posterior elongation in the intermediate-germband cricket Gryllus bimaculatus. Mechanisms of Development 122, 231–239 Shinmyo et al. (unpublished data) observed the effects of paRNAi on caudal development in Gryllus embryos where only the head region is formed in eggs after the injection of dsRNA targeting Gb’caudal in female crickets. Bucher et al.(2002) found that parental RNAi was highly efficient in Tribolium  Nearly 100% of embryos in the first egg laid after injection had RNAi phenotypes. 13
    • 14. Large numbers of RNAi embryos can be readily obtained with paRNAi Crucial for many insects whose eggs are not accessible or do not survive with microinjection (Bucher et al.2002). Investigations in the field of evolutionary developmental biology Why Parental RNAi? From Gregor Bucher, Johannes Scholten, Martin Klingle. (2002) Parental RNAi in Tribolium (Coleoptera) Current biology ,Volume 12, Issue 3, R85–R86 14
    • 15. Embryonic RNAi RNAi construct targeting the gene of interest is injected into developing eggs From Grossmann D, Scholten J, Prpic NM (2009) Separable functions of wingless in distal and ventral patterning of the Tribolium leg. Development Genes and Evolution 219, 469–479 Grossmann et al. (2009) found that paRNAi experiments lead to either empty egg shells or wild-type cuticles. emRNAi was able to circumvent the problem of effects exhibited during gonad formation or oogenesis.  The effects that lead to early embryonic lethality was also excluded. Why Embryonic RNAi? 15
    • 16. Larval/nymphal /pupal RNAi Analyze gene functions in postembryonic development to study the molecular basis of adult morphological diversity in various organisms.  Useful for analyzing the functions of enzymes in biosynthesis pathways. Nymphal RNAi (nyRNAi) was reported by Dong and Friedrich (2005) as a systemic RNAi mediated gene knockdown in the juvenile grasshopper, Schistocerca americana. From G. bimaculatus. Bando et al. (unpublished results) 16
    • 17. Regeneration-dependent RNAi Nymphs of hemimetabolous insects, such as cockroaches and crickets, exhibit a remarkable capacity for regenerating complex structures from damaged legs  No phenotype is induced by nyRNAi in an intact cricket leg  Bando et al. (2009) used a cricket leg model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. From Bando T, Mito T, Maeda Y et al. (2009) Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136, 2235–2245 17
    • 18. APPLICATIONS OF RNAi FOR DEVELOPMENT OF SPECIES-SPECIFIC dsRNA INSECTICIDES Pros: Provides an environmental friendly approach to generate plants resistant to viruses and insects kill only the target animals by down-regulating essential gene functions Cons: Relies on the injection of dsRNA into insects, which is not possible for practical application of insecticides. 18
    • 19. EFFICIENCY OF RNAI DEPENDS ON DEVELOPMENTAL STAGES AND SPECIES The dependence of RNAi efficiency on developmental stages or species is attributed to Differences in the expression patterns of the sid orthologs (C.elegans systemic RNAi defective mutant gene )  Differences in the sensitivity of dsRNA detection systems for endocytosis in insect cells 19
    • 20. FUTURE PROSPECTS FOR RNAI-BASED EXPERIMENTS IN INSECTS The mechanisms underlying RNAi phenomena, such as species or tissue dependent changes in the sensitivity to dsRNAs, are not fully understood. Systemic RNAi-based genome-wide screening is particularly useful to identify genes involved at the whole animal level, The insect world, with its more than 30 orders, about 1 million described species, and perhaps 30 million still awaiting taxonomic discovery, still hides many genetic black boxes, and RNAi appears to be a formidable key for opening a large number of them. 20
    • 21. Thank You……..Any questions? 21

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