Systemic RNAi Research Articles

SID-1 protein of Caenorhabditis elegans mediates uptake of dsRNA into Bombyx cells

RNA interference is one of the most revolutionary tools in the study of gene function, particularly in non-model systems. However, in Bombyx mori, as with many lepidopteran species, attempts at systemic RNAi have had mixed success. Gene identification and phylogenetic analyses suggest that Bombyx has the core RNAi machinery, which is necessary to undergo RNAi as a cellular response. We introduced sid genes from Caenorhabditis elegans into Bombyx BmN4 cells to enhance the uptake of dsRNA and revealed that the SID-1 protein, but not SID-2, has the ability to endow the RNAi effect with the addition of dsRNA to the medium. Observed RNAi effect was dependent on both the levels of sid-1 expression and the concentration of the dsRNA. These results suggest that SID-1 promotes the uptake of dsRNA from the medium into Bombyx cells. We generated transgenic animals that express sid-1 but have not detected significant enhancements of in vivo phenotype in response to the injection of the dsRNA into hemocoel.

Scavenger Receptor Mediates Systemic RNA Interference in Ticks

RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.

RNA interference inCaenorhabditis elegans: Uptake, mechanism, and regulation

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode <em>Pristionchus pacificus</em>

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F(1) progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode <em>Pristionchus pacificus</em>

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor

A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.

Abstract 4441: Intracellular delivery of therapeutic siRNA via an antennapedia-RNA-binding domain fusion peptide

Abstract A key characteristic in tumorigenesis is the aberrant expression of various oncogenes, whose cellular products drive various anti-apoptotic/pro-survival pathways. The RNAi pathway is an endogenous cellular pathway that transiently inhibits the translation of mRNA into protein. It therefore has the potential to selectively arrest the production of mutated proteins. As the Intracellular delivery of bioactive molecules is still a major challenge, we are exploring the possible ability of the Antenapedia homeodomain (Antp) to act as an effective transporter of conjugated cargo across biological membranes. We have previously shown that Antp delivers both p21 into p21-null tumors in order to restore cell-cycle regulation, as well as a truncated form of MAML to inhibit Notch activation and induce apoptosis. To test whether in silico-designed siRNA has the ability to silence mutated forms of genes, we have created a bifunctional fusion protein, TR7, which incorporates Antp and an RNA-binding protein. TR7 attempts to both neutralize the anionic nature of siRNA as well as introduce it to the cytoplasm, thus initiating the RNAi process in a non-cytotoxic manner. In summary, this project, utilizing Antp, aims to solve the issue of systemic RNAi delivery, as well as harness the endogenous RNAi pathway to effectively halt the production of abnormal proteins in cancer and other life threatening illnesses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2011-4441

SID-1 is a dsRNA-selective dsRNA-gated channel

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.

Systemic RNA-interference in the honeybee Apis mellifera: Tissue dependent uptake of fluorescent siRNA after intra-abdominal application observed by laser-scanning microscopy

RNA interference has been successfully used in adult honeybees, but there are only few reports about abdominal application of dsRNA/siRNA which have reached more distant tissues than the fat body. We studied systemic RNAi in honeybees by injecting fluorescent siRNA of the ubiquitously expressed honeybee homologue of the Glycerol-3-Phosphate Dehydrogenase (amGpdh) into the abdomens of adult bees and followed them by laser scanning microscopy and qPCR. The fat body was the sole tissue emitting fluorescence and showing a decreased gene expression, whereas the siRNA had apparently not reached the other tissues. Therefore, we conclude that certain genes in other tissues than the fat body cannot be easily reached by injecting siRNA into the body cavity. In particular, the lack of amGpdh knock down in ovaries after amGpdh dsRNA injection, supports that in some cases it may be particularly difficult to interfere with gene expression in ovaries by intra-abdominal injection. In these cases alternative inhibition techniques may be required to achieve an organismic non-lethal disruption of transcription.

PTP1B Suppresses Prolactin Activation of Stat5 in Breast Cancer Cells

Basal levels of nuclear localized, tyrosine phosphorylated Stat5 are present in healthy human breast epithelia. In contrast, Stat5 phosphorylation is frequently lost during breast cancer progression, a finding that correlates with loss of histological differentiation and poor patient prognosis. Identifying the mechanisms underlying loss of Stat5 phosphorylation could provide novel targets for breast cancer therapy. Pervanadate, a general tyrosine phosphatase inhibitor, revealed marked phosphatase regulation of Stat5 activity in breast cancer cells. Lentiviral-mediated shRNA allowed specific examination of the regulatory role of five tyrosine phosphatases (PTP1B, TC-PTP, SHP1, SHP2, and VHR), previously implicated in Stat5 regulation in various systems. Enhanced and sustained prolactin-induced Stat5 tyrosine phosphorylation was observed in T47D and MCF7 breast cancer cells selectively in response to PTP1B depletion. Conversely, PTP1B overexpression suppressed prolactin-induced Stat5 tyrosine phosphorylation. Furthermore, PTP1B knockdown increased Stat5 reporter gene activity. Mechanistically, PTP1B suppression of Stat5 phosphorylation was mediated, at least in part, through inhibitory dephosphorylation of the Stat5 tyrosine kinase, Jak2. PTP1B knockdown enhanced sensitivity of T47D cells to prolactin phosphorylation of Stat5 by reducing the EC(50) from 7.2 nmol/L to 2.5 nmol/L. Immunohistochemical analyses of two independent clinical breast cancer materials revealed significant negative correlations between levels of active Stat5 and PTP1B, but not TC-PTP. Collectively, our data implicate PTP1B as an important negative regulator of Stat5 phosphorylation in invasive breast cancer.

Enforcing biphasic eye development in a directly developing insect by transient knockdown of single eye selector genes

The visual system of indirectly developing insects such as Drosophila passes through two phases of development. Larval eyes form in the embryo, whereas the adult compound eyes form during metamorphosis. Comparative evidence implies that this biphasic mode of visual system development evolved from the continuously developing eye of directly developing insects. We investigated the developmental basis of this evolutionary transformation in a directly developing insect taking advantage of the time-limited nature of systemic RNAi in the grasshopper Schistocerca americana. Transient knockdown of the homologs of the early retinal genes eyes absent (eya) or sine oculis (so) both induced long-term arrest of eye development in grasshopper nymphs. Eye development, however, resumed after knockdown expiry. This finding sheds first light on the molecular regulation of postembryonic eye development in directly developing insects and unravels an inherent capacity of the underlying gene regulatory network to accommodate for partitioning visual system development into discrete phases, as in indirectly developing insects.

Nanotubes Functionalized with Lipids and Natural Amino Acid Dendrimers: A New Strategy to Create Nanomaterials for Delivering Systemic RNAi

Single-walled carbon nanotubes (SWNT) have unique electronic, mechanical, and structural properties as well as chemical stability that make them ideal nanomaterials for applications in materials science and medicine. Here, we report the design and creation of a novel strategy for functionalizing SWNT to systemically silence a target gene in mice by delivering siRNA at doses of <1 mg/kg. SWNT were functionalized with lipids and natural amino acid-based dendrimers (TOT) and complexed to siRNA. Our model study of the silencing efficiency of the TOT-siRNA complex showed that, in mice injected at 0.96 mg/kg, an endogenous gene for apoliproprotein B (ApoB) was silenced in liver, plasma levels of ApoB decreased, and total plasma cholesterol decreased. TOT-siRNA treatment was nontoxic and did not induce an immune response. Most (80%) of the RNA trigger molecules assembled with TOT were cleared from the body 48 h after injection, suggesting that the nanotubes did not cause siRNA aggregation or inhibit biodegradation and drug clearance in vivo. These results provide the first evidence that nanotubes can be functionalized with lipids and amino acids to systemically deliver siRNA. This new technology not only can be used for systemic RNAi, but may also be used to deliver other drugs in vivo.

Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages ( TcCTLP-5A/ B, TcCTLP-6A), others were also detected in non-feeding embryonic ( TcCTLP-5C, TcCTLP-6D) and pupal stages ( TcCTLP-5C, TcCTLP-6C/ D/ E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects.

Target of Rapamycin (TOR) Mediates the Transduction of Nutritional Signals into Juvenile Hormone Production

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

TheCaenorhabditis elegans rsd-2andrsd-6Genes Are Required for Chromosome Functions During Exposure to Unfavorable Environments

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.

Cloning and Phylogenetic Analysis of Sid-1-Like Genes from Aphids

The sid-1 (systemic interference defective) gene encodes a transmembrane protein that is an important participator in the systemic RNAi pathway and has been reported in several organisms. In insects, sid-1-like genes were described from Tribolium castaneum, Apis mellifera, Bombyx mori and Schistocerca americana, but were not found in Drosophila melanogaster and Anopheles gambiae. To investigate whether this gene occurs in aphid species, RT-PCRs were performed using degenerate primers designed using the conserved motif of sid-1-like genes. An sid-1-like full-length transcript was amplified from the cotton/melon aphid, Aphis gossypii Glover (Homopera: Aphididae), and a fragment was amplified from the grain aphid, Sitobion avenae (F.). The trancript from A. gossypii was 3067 bp long, with an open reading frame encoding 766 amino acids. Sequence analysis indicated that this transcript shares highest similarity with the reported sid-1-like gene in Schistocerca americana (53%, fragment), followed by A. mellifera (44%), T. castaneum (32–44%), B. mori (38–42%) and Caenorhabditis elegans (25%). Analysis of the transmembrane protein topological structure indicated that the protein encoded by this gene has a similar structure to SID-1 of C. elegans. A phylogenetic tree with all available sid-1-like genes suggests that sid-1-like genes may have had a long evolutionary history. Considering its importance in the RNAi pathway, the absence of a sid-1-like gene in D. melanogaster and A. gambiae is worthy of further investigation.

Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

Tribolium resembles C. elegans in showing a robust systemic RNAi response, but does not have C. elegans-type RNAi mechanisms; insect systemic RNAi probably uses a different mechanism.

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.

Functions of the ecdysone receptor isoform-A in the hemimetabolous insect Blattella germanica revealed by systemic RNAi in vivo

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we show that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, we reduced its expression using systemic RNAi in vivo, and we obtained knockdown specimens. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis.

Breakthrough for systemic RNAi

Breakthrough for systemic RNAi