Use of piggyBac and a Sindbis Virus for Foreign Gene Expression and RNAi in the Silkworm

Jindra M, Uhlirova M.

Publication Details

Research on insects cannot advance without developing efficient methods for gene transfer, forced expression and silencing in vivo. Here we present recent developments of heat-inducible gene misexpression based on the piggyBac transposon, and a recombinant Sindbis virus as a novel means of endogenous gene silencing in the silkworm. Although both systems have their shortcomings, they will permit functional genetic studies in nonmodel insects.

Reverse Genetics in Drosophila and the Silkworm

Genetic research on nondrosophilid insects is important for two reasons. First, future management of pests and disease vectors will probably rely on transgenic technologies. Second, our understanding of general evolutionary-developmental (evo-devo) mechanisms will require studies on more than one species, the fruit fly Drosophila melanogaster. The fly has served for decades as an excellent model for functional genetics. Much of its success is owing to sophisticated techniques of reverse genetics, based on germline transformation with the transposable element P. We will first review the transgenic tools available for Drosophila and then discuss development of similar tools for studies in nondrosophilid species, represented here by the silkworm Bombyx mori.

The Drosophila Transgenic Toolbox

The P element-mediated transformation of Drosophila melanogaster, pioneered in 1982 by Rubin and Spradling,1 revolutionized genetic research and contributed to making the fly an important model organism. Engineered P elements lacking their autonomous ability to transpose can be stably integrated into the Drosophila genome and remobilized simply by crossing the P element-carrying fly with one possessing a transgenic transposase activity. This trick is often used for mutagenesis, since the mobilized P can insert into random chromosomal loci.2 New gene regulatory elements can be discovered using a variation on this method, known as enhancer trap.3 In this case the P element carries a reporter gene, e.g., for the enzyme β-galactosidase, whose activity becomes visible upon insertion of the P near an endogenous transcriptional enhancer.

One important facility of the Drosophila transformation system is ectopic targeting of a gene expression to tissues and at times of choice. This ability helps us define where and when the normal function of a given gene is required for development. For instance, expression of a gene targeted to the epidermis but not to the gut may be sufficient to rescue mutants lacking that gene.4 Alternatively, misexpression of a protein at the wrong time or tissue may cause an aberrant phenotype, revealing a function of the gene.5 Targeted overexpression of dominant-negative protein forms that compete in vivo with the natural gene products can also be instructive.4,6 Finally, transgenic expression of double-stranded RNA can bring about gene-specific post-transcriptional silencing and thus substitute for tedious mutagenesis. This “knockdown” technique relies on the process known as RNA interference (RNAi), whereby double-stranded RNA triggers an enzymatic degradation of the homologous, endogenous mRNA, and thus produces loss-of-function phenotypes for the targeted gene.7-9 The availability of tissue-specific or inducible regulatory cis-elements (promoters and enhancers) in Drosophila allows RNAi to be restricted to particular tissues and developmental times. Such conditional knockdowns are important to reveal late developmental functions of genes, whose loss is lethal at early stages. For example, RNAi induced in Drosophila larvae has shown that two steroid response proteins, EcR and Ftz-F1, are required for metamorphosis, even though mutants lacking either one of these factors would die as embryos.9

Conditional or temporally restricted protein or RNA expression is most commonly achieved using a heat-inducible promoter from the Drosophila heat-shock protein 70 (hsp70) gene. A portion of the hsp70 promoter, sufficient for the heat response, is cloned within one P element vector upstream of the sequence to be expressed.10 One-hour exposure of larvae, pupae or flies carrying such a construct to 37-38°C causes overexpression of the transgene. RNA and protein levels culminate soon after the heat shock and then decline within several hours. Although convenient, the heat-inducible overexpression system has its problems that may hamper some experiments. Not only heat but also other insults activate the hsp70 promoter, which confers basal expression in the absence of a heat treatment. Moreover, temperatures near 37°C are not tolerated without side effects. Levels of heat-induced expression vary among individual transgenic lines due to position effects on the P element construct. Finally, the ectopic hsp70-dependent expression occurs in most tissue types, thus affecting the whole animal.

An efficient way of targeted gene expression uses a binary system, based on the yeast transcription factor GAL4 and its cognate cis-element, the Upstream Activation Sequence (UAS).11 This method requires two strains of transgenic flies: a responder in which the gene of interest is cloned behind the UAS, and a driver expressing GAL4 under the desired tissue-specific promoter. When such driver and responder flies are crossed, binding of GAL4 to the UAS causes a strong expression of the UAS-fused gene in their progeny. The expression only occurs in tissues where the GAL4-driving enhancer is active, thus producing local protein overexpression or RNAi knockdown. This system can also be used for heat-inducible expression when a UAS responder strain is crossed with a driver that carries GAL4 under the hsp70 promoter. However, it should be noted that high abundance of the GAL4 protein does affect Drosophila development.

Silkworm As a Model for Developmental Genetics

With the powerful Drosophila system in hand, any additional insect model might seem redundant. However, some aspects of insect biology, such as plant-herbivore or host-parasite interactions, pathogen transfer, seasonal developmental rhythms or silk production, need to be addressed directly in the relevant species.12 Although Drosophila continues to be an excellent model for deciphering the basic principles of animal development, some of the findings may not apply to insects universally. In fact, they might reflect exceptional features of the highly evolved fly species.

Metamorphosis and its hormonal control is one of the developmental processes worth studying in alternative models such as the silkworm. The reason is that during metamorphosis of the higher flies, the external adult structure is built from groups of primordial cells that replace the existing larval epidermis: the entire head and thorax are made from imaginal discs and the abdomen from nests of histoblasts.13 By contrast, only the silkmoth wings originate from true imaginal discs, while appendages such as the antennae and legs grow from precursor cells that are until metamorphosis buried inside of the functional larval organs.14 However, most of the Bombyx larval epidermis is not replaced but is reprogrammed to produce first the pupal and then the adult cuticle, as is true for other typical holometabolous insects.15,16 Despite these different strategies, metamorphosis in both flies and moths is governed by the same steroid hormone ecdysone, acting through a pathway of molecularly well-conserved genes.17,18 Only loss-of-function studies on insects other than Drosophila can show whether these genes also have conserved developmental roles.

Since the P element can only be used in drosophilids, reverse genetics in other insects has been hindered until the findings of transposable elements with a broader host range (reviewed in refs. 19-21). Among these transposons, piggyBac has a good potential as a vector for reverse genetic applications.12,22 piggyBac is thus far the only transposon reported for germline transformation of lepidopterans, namely Bombyx mori23-25 and the pink bollworm Pectinophora gossypiella.26 The use of piggyBac was greatly facilitated by the addition of a strong EGFP marker, driven by binding sites (3xP3) for the eye-specific transcription factor Pax6.27-29 Chapters in this book document that silkworm transformation with piggyBac has become routine.

Putting a foreign gene into the host genome, however, is only one step en route to reverse genetic studies. The other problem is how to drive the transgene expression where and when desired. Besides the artificial “promoter” 3xP324,25 only very few promoters have thus far been tested in live silkworms. These derive from a semi-ubiquitous cytoplasmic actin 3 gene (BmA3),23,30 a silk gland-specific fibroin light-chain,31,31 and a ubiquitous immediate early-1 (IE1) gene from the Bombyx nuclear polyhedrovirus BmNPV.32,33 Studies using the IE1 promoter in transgenic Bombyx have been thus far unsuccessful in yielding robust phenotypic effects. IE1-driven expression of an inhibitory version of the IE1 BmNPV protein was insufficient to render silkworms resistant to the virus.32 Similarly, misexpression of a female-specific Doublesex protein failed to feminize Bombyx males, although it upregulated a yolk gene in the male fat body.33 None of these promoters, however, can be induced at will. To achieve an inducible transgene expression, we have equipped piggyBac with the Drosophila hsp70 promoter.25 As will be discussed in the following section, also heat-induced misexpression or RNAi of an essential gene ftz-f1 could not elicit aberrant phenotypes in the silkworm.

These problems are likely due to the poor selection of promoters that would ensure massive overexpression. A possible remedy is an implementation of the GAL4/UAS system, whereby ectopic expression can be amplified by the GAL4 activity. Imamura and colleagues recently demonstrated that GAL4/UAS can target gene expression in Bombyx.30 Yet, this approach alone does not solve the problem of promoter shortage, because one still needs something to drive the expression of GAL4.

Heat-Inducible Gene Expression Using piggyBac Transgenic Constructs

Our goal was to misexpress or knockdown in Bombyx proteins of the ecdysone signaling pathway, such as the nuclear receptor Ftz-F1. Mutant and ectopic expression studies in Drosophila have shown that Ftz-F1 is required for embryogenesis, then during the larval life for molting, and eventually for pupation and metamorphosis.34,35 Therefore we needed a conditional expression that could interfere with the postembryonic developmental events. As discussed above, one suitable promoter for inducible transgenic expression is hsp70. We had two reasons to believe that hsp70 is a good choice. First, lepidopteran larvae have been known to respond to warming by expressing heat shock proteins36 and the Drosophila hsp70 promoter was active in two lepidopteran cell lines,37,38 thus encouraging the idea that it might work also in live silkworms. Second, when driven in Drosophila by hsp70, premature Ftz-F1 expression was lethal35 and Ftz-F1 RNAi9 produced phenocopies of ftz-f1 mutations.

Vector Design and Bombyx Transformation

We constructed piggyBac vectors pBac{hsFtz-F1} and pBac{hsFtz-F1RNAi} based on the original pBac{3xP3-EGFPafm} of Horn and Wimmer.29 Sequences of ftz-f1 to be expressed were placed between the Drosophila hsp70 promoter and the terminator.25 pBac{hsFtz-F1} contained the entire ftz-f1 coding sequence (fig.1). pBac{hsFtz-F1RNAi} designed for hairpin-loop dsRNA expression contained an inverted repeat of the same 820-bp ftz-f1 cDNA fragment, separated by an intron (fig.1) that should facilitate RNAi.39

Figure 1. Design of piggyBacvectors pBac{hsFtz-F1} and pBac{hsFtz-F1RNAi} for heat-inducible overexpression and RNAi knockdown of the Ftz-F1 protein.

Figure 1

Design of piggyBacvectors pBac{hsFtz-F1} and pBac{hsFtz-F1RNAi} for heat-inducible overexpression and RNAi knockdown of the Ftz-F1 protein. Arrowheads denote primers used for RT-PCR detection of the transgenic mRNA.

Three independent transgenic lines were established for pBac{hsFtz-F1} and 10 for pBac{hsFtz-F1RNAi} using transformation of the silkworm strain Nistari as described.25 These 13 plus 12 other transgenic lines were produced in our laboratory over two years with average efficiencies around 5% of G0 adults that yielded transformed progenies. DNA injections were done on embryos within 90 minutes after egg laying using a manual micromanipulator and only a glass needle, prepared from Narishige GDC-1 capillaries on a vertical needle puller. The position of the injection point (mid-ventral) and keeping embryos in moderate humidity after injection and sealing with an acrylic glue appeared to be the most critical conditions for successful transformation.

All lines that we tested by using inverse PCR showed chromosomal integration of the piggyBac constructs, generally a single insertion. The complete cassettes for protein expression were always present in the Bombyx genomic DNA, parts of the RNAi constructs however were lost in four of the pBac{hsFtz-F1RNAi} lines, probably due to a recombination between the long ftz-f1 inverted repeats.

Tissue and Temporal Patterns of Heat-Inducible Expression

Several transgenic lines carrying the hsp70 cassette were tested for inducibility of the RNA expression by using PCR primers specific to the transgene (fig.1) and on Northern blots. Animals were exposed to a heat shock for 1 hour or 90 minutes. Larvae were heated either individually in tubes submersed in a water bath or in groups placed in an air oven; embryos and pupae were heated only in the oven. In summary, 42°C was required for a substantial induction at any stage tested; only a weak RNA increase was seen at 40°C, and no induction occurred after 90 minutes at 38°C. Thus the temperature required corresponded to that causing expression of heat shock proteins in live lepidopterans.36

The transgene was inducible already in day 1 embryos and then until hatching (fig.2A). Heat shock-independent basal hsp70 activity was observed in embryos and in larval tissues. Quantitative RT-PCR in the larval epidermis showed that the transgenic ftz-f1 mRNA expression culminated 1 hour after heat shock at a 30-fold increase over the basal level, then it declined by 16 hours post treatment (fig.2B). At the 3-hour point, increased levels of the Ftz-F1 protein were detected in the epidermal nuclei. A higher fold increase was found in the posterior silk glands (fig.2C), partly because the basal hsp70 activity appeared lower, and probably also because of the enormous transcriptional potential of this highly polyploid organ. A super-induction by another heat shock further increased the mRNA to over a 100-fold the uninduced level (fig.2C).

Figure 2. Induction of the transgenic ftz-f1 products using the pBac{hsFtz-F1} construct.

Figure 2

Induction of the transgenic ftz-f1 products using the pBac{hsFtz-F1} construct. Embryos or day 1 fifth-instar silkworms were kept at 42°C for 60 min and then whole embryos (A) or larval organs were analyzed 1 hour after the heat shock unless otherwise (more...)

Interestingly, the mRNA could also be ectopically induced in larval gonads, particularly the testes (fig.2D). This suggests a possibility to enhance germline transformation by using an hsp70-driven transposase helper. Although such a helper plasmid was efficient for transformation of the pink bollworm,26 there are no reports on its use in Bombyx.

Disadvantages of the System

The obvious problem of the hsp70 system is the high temperature required for induction. Heat shocks at 42°C disrupt development when applied at some stages. Particularly sensitive are embryos at around 70% of development and larvae during all molts; younger embryos, feeding larvae, pupae and adults tolerate heating quite well.

Even though we tested 10 lines with the potential of Ftz-F1 knockdown, we failed to obtain specific loss-of-function phenotypes. It is possible that even if such phenotypes occurred, they might have been obscured by defects caused by heat alone. Unfortunately, heat shocks disrupt molting and pupation, i.e., defects anticipated for the loss of ftz-f1 based on studies in Drosophila.9,34,35 One problem is that while a heat-induced expression lasts for only a small fraction of development in Bombyx, in Drosophila it represents a significant period. Most likely however the expression levels conferred by the hsp70 promoter are too low to trigger efficient RNAi. The approach may require more potent promoters, perhaps linked to the GAL4/UAS system.

Use of Sindbis Viruses for Protein Misexpression and RNAi in Bombyx

Two major problems are encountered with transgenic applications based on the piggyBac transformation in the silkworm. First, the approach is inherently slow with minimum 45 days per generation and months before a transgenic line may be established. Second, the current lack of a variety of strong specific enhancers does not permit massive overexpression that would confer a dominant-negative effect of a protein, or an RNAi knock-down.

RNA viruses whose expression is independent of the nuclear transcription provide an alternative solution. The alphavirus Sindbis (SIN) appears particularly suitable as a vector for gene manipulations in insects, since its infection is noncytopathic in a broad range of hosts. Engineered double-subgenomic Sindbis (dsSIN) viruses have been successfully used to force gene expression in mosquitoes.40-42 dsSIN-mediated misexpression of the Drosophila gene Ultrabithorax produced expected homeotic transformations in the butterfly Precis coenia and the beetle Tribolium castaneum, thus demonstrating a great promise of the system for evo-devo genetic studies.43 Although there are lepidopterans, such as the tobacco hornworm Manduca sexta, that resist SIN infection,44 we have established that Bombyx mori is a susceptible species.44,45

An attractive possibility is to employ dsSIN for loss-of-function studies using RNAi. Sindbis has a positive strand RNA genome, from which nonstructural genes are translated on host cell ribosomes immediately upon infection. A viral RNA-dependent RNA polymerase then produces a negative sense RNA copy of the genome, which serves as a template for synthesis of first and second subgenomic RNAs and also to make new positive RNA strand (fig.3A). This led to the idea that a heterologous sequence, inserted into a replicating dsSIN genome in either direction, will produce a double-stranded RNA that will in turn trigger RNAi knockdown of the inserted gene. Indeed, such recombinant viruses silenced transgenic and endogenous mosquito genes46,47 or reduced the competence of Aedes aegypti to transmit the dengue virus.48 Recently a knockdown of a GATA transcription factor was achieved in the same mosquito species by expressing a hairpin-loop GATA RNA double-strand from a construct placed behind the second subgenomic dsSIN promoter.49 The RNAi mechanism is marked by the appearance of gene-specific small interfering RNAs (siRNAs) that mediate the RNA degradation into 21-23-nt pieces.7 Upon a dsSIN-mediated knockdown in Bombyx, we have detected such gene-specific siRNAs in the tissues infected by the recombinant virus (fig.3B), thus demonstrating that transgenic RNAi is functional in the silkworm.45

Figure 3. Sindbis virus-mediated RNAi of BR-C.

Figure 3

Sindbis virus-mediated RNAi of BR-C. (A) Recombinant TE 3'2J-based virus designed for BR-C RNAi carries a 14-kb positive strand RNA genome with a second subgenomic promoter at the 3' end, where the 705-bp BR-C cDNA fragment was cloned in antisense orientation. (more...)

RNAi Reveals That BR-C Is Required for Metamorphosis in Bombyx

We chose to target the Broad-Complex (BR-C) protein because of its critical role in Drosophila metamorphosis.50 Unlike other ecdysone-induced transcription factors, BR-C is not required for postembryonic development until pupation, for which it appears to be a master switch.16 We prepared a BR-CRNAi dsSIN by cloning a 700-bp BR-C cDNA fragment in the antisense orientation behind the second subgenomic promoter of a TE 3'2J SIN virus (fig.3A). A control expressing EGFP in place of the BR-C RNA was used to monitor the spreading of the virus in vivo. Finally, a fusion of the EGFP coding region and the antisense BR-C fragment was designed to mark tissues in which RNAi took place. Infectious viruses were prepared from the plasmid DNA constructs in three steps: (1) in vitro transcription and capping of the viral genome, (2) electroporation of hamster BHK-21 cells with this RNA, and (3) harvesting cell culture supernatant containing viral particles. Viruses with titers around 106 pfu/ml were injected between abdominal segments of mid-fourth or early-fifth instar silkworms.45

Larval organs such as the fat body and silk glands, and precursors of imaginal structures were infected by dsSIN.44 Most larvae injected with the control EGFP virus produced adults with EGFP+ eyes, antennae, legs and wings, but with no profound morphological defects. Infection with both BR-CRNAi constructs disrupted metamorphosis in several ways.45 Briefly, infection of fourth-instar larvae blocked pupation in over 50% of the animals (fig.3D); those which pupated failed to extend wings and legs, and usually died. Leg and wing formation defects were observed in pupae (fig.3F) and also in adults when fifth-instar larvae were infected. In addition, these adults displayed malformed compound eyes (fig.3I). Besides differentiation of adult structures, metamorphosis also involves degeneration of obsolete larval organs. BR-C RNAi prevented the programmed death of Bombyx silk glands that normally occurs in early pupae. All of these defects correspond well with the morphogenetic and degenerative aspects of BR-C function during Drosophila metamorphosis, revealed by mutant studies.50-52

Drawbacks of the dsSIN system include restricted tissue tropism. Since some Bombyx organs such as the larval epidermis and the gonads apparently resist SIN infection, the animals treated with SIN must be regarded as genetic mosaics. Experiments that require heritable transformation are not practical with Sindbis. There is also a limitation as to the maximal lengths of RNA that may be added to the viral genome; dsSIN tends to delete long inserts placed at its 3' end. Finally, the virus infects mammalian cells, in which it is usually propagated, and thus SIN infection may be harmful to humans.

Conclusions and Future Perspective

The heat-inducible piggyBac vector based on the Drosophila hsp70 promoter failed to produce misexpression defects or RNAi knockdown of the ftz-f1 gene in Bombyx, however, for the first time it allowed a temporal control over foreign gene expression in a live nondrosophilid insect. Although not effective for ftz-f1, the system may enable studies on other genes. Clearly, better inducible vectors, such as those employing the Tetracycline repressor, should be developed. Future applications will also require expanding the list of available tissue-specific enhancers that could be used in conjunction with the GAL4/UAS system. A strategy for finding such enhancers using piggyBac in nondrosophilid insects has been recently outlined.12,22 Recombinant Sindbis viruses provide a fast and potent alternative for ectopic expression and RNAi in the silkworm. Together, the viral and transposon-based vectors will in near future enable functional genetic studies in Bombyx and other species.

Acknowledgments

We wish to thank our collaborators of the Ken Olson and Barry Beaty group at the AIDL, Fort Collins, CO and to Masako Asahina and Lynn Riddiford, who collaborated with us on the FIRCA NIH grant R03 TWO1209-01 that partly supported this work. Other support was a grant IAA5007305 from the Czech Academy of Sciences to MJ and a NATO Science Fellowship to MU.

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