Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/<Emphasis Type="Italic">FRT</Emphasis> site-specific recombination system

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.     

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.     

Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells

To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700—800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×10⁵ cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.     

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.     

A new method for the modification of fibroin heavy chain protein in the transgenic silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.     

A transgenic silkworm expressing the immune-inducible cecropin B-GFP reporter gene

To analyze cecropin B promoter (P-CecB) activity in vivo, we constructed transgenic silkworms that expressed EGFP under the control of P-CecB using the piggyBac transposable element. Genomic Southern blot analysis of the G1 and G2 generations indicated the stable insertion of EGFP in the genome. Injection of Escherichia coli cells into the larvae strongly induced EGFP expression in the fat bodies and all five hemocyte cell types. Northern blot analysis indicated that the expression kinetics of EGFP in the fat bodies following bacterial injection were correlated with that of endogenous CecB. Flow cytometric analysis of the hemocytes revealed that EGFP expression was increased by bacteria, but not by yeast. Our results indicate that the features of EGFP expression in the transgenic silkworm are equivalent to those of endogenous CecB and that P-CecB activation can be monitored by EGFP expression using transgenic silkworms.     

Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.     

A Novel Method to Convert a DNA Fragment Inserted into a Plasmid to an Inverted Repeat Structure

Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.     

转蜘蛛拖牵丝蛋白基因家蚕蚕丝氨基酸组成及其机械性能

将以绿色荧光蛋白基因为报告基因、含有人工合成的1.6 kb的蜘蛛拖牵丝蛋白基因及转座子pig-gyBac的转基因载体成功导入减秋无滞育家蚕受精卵,得到转蜘蛛拖牵丝蛋白基因家蚕及绿色荧光茧。对转基因家蚕与对照家蚕丝素蛋白进行了氨基酸组成分析,结果表明转基因蚕茧丝素蛋白甘氨酸和丙氨酸的百分含量分别增加了1.65%和1.80%(平均值);对其生丝的机械性能进行了测试研究,结果表明转基因蚕茧生丝的伸长率降低,断裂强度和初始模量增加,且差异均显著,与理论预期结果吻合。结果表明转基因家蚕蚕丝的机械性能一定程度上得到了提高。     

家蚕BmN细胞中8种启动子的活性比较及利用hr5-IE1启动子实现EGFP的稳定表达

为筛选出一个较强的启动子用于提高转座子piggyBac在家蚕Bombyx mori细胞中的转化效率,采用双荧光素酶报告基因检测（dual-luciferase reporter assay）技术比较了热激蛋白启动子（hsp70和hsp82）、家蚕肌动蛋白启动子（A3）、多聚泛素（polyubiquitin）启动子（PUB）、α微管蛋白启动子（α-tub）、丝素轻链启动子（Fib-L）、人工合成启动子3×P3及苜蓿丫纹夜蛾多角体病毒（AcNPV）增强子-启动子组合（hr5-IE1）8种启动子在家蚕细胞株BmN内的活性。结果显示hr5-IE1活性最强,A3次之,其余启动子活性均较弱。构建含有hr5-IE1启动子和piggyBac的转座酶编码区的质粒作为辅助质粒,与EGFP载体质粒一起转染家蚕细胞后,实现了EGFP基因整合到细胞基因组中。因此,今后可考虑将hr5-IE1用于家蚕细胞遗传转化的研究中,以提高细胞转化的效率。     

家蚕转基因载体pBacA3EG的构建及其表达

以家蚕Bombyx mori肌动蛋白A3(actin 3)启动子、增强性绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因及SV40的多聚腺苷酸识别序列为元件,经多次克隆,将其插入到piggyBac转座载体中。经PCR、酶切鉴定及测序表明各元件已按正确的方式插入到piggyBac载体中。将构建好的piggyBac表达载体显微注射到胚盘形成前期的蚕卵中,在胚胎早期发育的第3天,通过体视荧光显微镜检测到蚕卵内发出较强的绿色荧光。结果表明该载体构建正确且能在蚕卵中进行表达。家蚕转基因载体的体外瞬时表达不但是成功进行家蚕转基因所必需的第一步,而且其自身也可以应用于基因的功能研究,为家蚕后基因组研究奠定了基础。     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

A transgenic sensor strain for monitoring the RNAi pathway in the yellow fever mosquito, Aedes aegypti

The RNA interference pathway functions as an antiviral defense in invertebrates. In order to generate a phenotypic marker which "senses" the status of the RNAi pathway in Aedes aegypti, transgenic strains were developed to express EGFP and DsRED marker genes in the eye, as well as double-stranded RNA homologous to a portion of the EGFP gene. Transgenic "sensor" mosquitoes exhibited robust eye-specific DsRED expression with little EGFP, indicating RNAi-based silencing. Cloning and high-throughput sequencing of small RNAs confirmed that the inverted-repeat transgene was successfully processed into short-interfering RNAs by the mosquito RNAi pathway. When the A. aegypti homologues of the genes DCR-2 or AGO-2 were knocked down, a clear increase in EGFP fluorescence was observed in the mosquito eyes. Knockdown of DCR-2 was also associated with an increase in EGFP mRNA levels, as determined by Northern blot and real-time PCR. Knockdown of AGO-3, a gene involved in the germline-specific piRNA pathway, did not restore EGFP expression at either the mRNA or protein level. This transgenic sensor strain can now be used to identify other components of the mosquito RNAi pathway and has the potential to be used in the identification of arboviral suppressors of RNAi.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

Utilization of the Bombyx mori heat shock protein 70 promoter for screening transgenic silkworms

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time‐consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro‐injected into 3060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected, not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage, but also in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven‐day‐old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.     

New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements

We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.