Gene Transposon Research Articles

Tol2 gene trap integrations in the zebrafish amyloid precursor protein genes appa and aplp2 reveal accumulation of secreted APP at the embryonic veins

The single spanning transmembrane amyloid precursor protein (APP) and its proteolytic product, amyloid-beta (Ab) peptide, have been intensely studied due to their role in the pathogenesis of Alzheimer's disease. However, the biological role of the secreted ectodomain of APP, which is also generated by proteolytic cleavage, is less well understood. Here, we report Tol2 red fluorescent protein (RFP) transposon gene trap integrations in the zebrafish amyloid precursor protein a (appa) and amyloid precursor-like protein 2 (aplp2) genes. The transposon integrations are predicted to disrupt the appa and aplp2 genes to primarily produce secreted ectodomains of the corresponding proteins that are fused to RFP. Our results indicate the Appa-RFP and Aplp2 fusion proteins are likely secreted from the central nervous system and accumulate in the embryonic veins independent of blood flow. The zebrafish appa and aplp2 transposon insertion alleles will be useful for investigating the biological role of the secreted form of APP.

Tetracycline resistance genes of ureaplasmas

In the medical setting, limited information is available on antimicrobial susceptibilities and resistance development in ureaplasmas. This study addresses tetracycline and doxycycline resistance in clinical isolates of Ureaplasma parvum and Ureaplasma urealyticum. Culture, with species polymerase chain reaction (PCR) confrmatory techniques, was applied to 191 endocervical specimens, collected during the period January-March 2006. Minimum inhibitory concentration (MIC) determinations were performed by microbroth dilution, with tetracycline resistance (tetM), and int-Tn genes were characterised by PCR and sequencing. Sixty-six ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum and U. urealyticum) were obtained. On screening the ureaplasma cultures, tetM gene regions were demonstrated from tetracycline-susceptible and -resistant ureaplasmas. Seven isolates (six U. parvum and one U. urealyticum) were resistant to tetracycline, with dual doxycycline resistance observed in three strains. Int-Tn gene characterisation of the seven tetracycline-resistant strains revealed that three types were present, indicating that transposons from different origins had integrated into ureaplasma genomes. TetM sequences of fve tetracycline-resistant strains were seen to be highly mosaic in structure. The fnding of transposon and/or tetM regions in all ureaplasma cultures investigated, with, or without, full expression of tetracycline resistance, in conjunction with tetM and int-Tn gene mosaic or diversity, verifes that ureaplasmas undergo extensive genetic exchange of transposon or resistance genes, with concomitant genomic remodelling.

Influence of Soil Use on Prevalence of Tetracycline, Streptomycin, and Erythromycin Resistance and Associated Resistance Genes

This study examined differences in antibiotic-resistant soil bacteria and the presence and quantity of resistance genes in soils with a range of management histories. We analyzed four soils from agricultural systems that were amended with manure from animals treated with erythromycin and exposed to streptomycin and/or oxytetracycline, as well as non-manure-amended compost and forest soil. Low concentrations of certain antibiotic resistance genes were detected using multiplex quantitative real-time PCR (qPCR), with tet(B), aad(A), and str(A) each present in only one soil and tet(M) and tet(W) detected in all soils. The most frequently detected resistance genes were tet(B), tet(D), tet(O), tet(T), and tet(W) for tetracycline resistance, str(A), str(B), and aac for streptomycin resistance, and erm(C), erm(V), erm(X), msr(A), ole(B), and vga for erythromycin resistance. Transposon genes specific for Tn916, Tn1549, TnB1230, Tn4451, and Tn5397 were detected in soil bacterial isolates. The MIC ranges of isolated bacteria for tetracycline, streptomycin, and erythromycin were 8 to >256 μg/ml, 6 to >1,024 μg/ml, and 0.094 to >256 μg/ml, respectively. Based on 16S rRNA gene similarity, isolated bacteria showed high sequence identity to genera typical of soil communities. Bacteria with the highest MICs were detected in manure-amended soils or soils from agricultural systems with a history of antibiotic use. Non-manure-amended soils yielded larger proportions of antibiotic-resistant bacteria, but these had lower MICs, carried fewer antibiotic resistance genes, and did not display multidrug resistance (MDR).

The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools

The Arabidopsis Information Resource (TAIR, http://arabidopsis.org) is a genome database for Arabidopsis thaliana, an important reference organism for many fundamental aspects of biology as well as basic and applied plant biology research. TAIR serves as a central access point for Arabidopsis data, annotates gene function and expression patterns using controlled vocabulary terms, and maintains and updates the A. thaliana genome assembly and annotation. TAIR also provides researchers with an extensive set of visualization and analysis tools. Recent developments include several new genome releases (TAIR8, TAIR9 and TAIR10) in which the A. thaliana assembly was updated, pseudogenes and transposon genes were re-annotated, and new data from proteomics and next generation transcriptome sequencing were incorporated into gene models and splice variants. Other highlights include progress on functional annotation of the genome and the release of several new tools including Textpresso for Arabidopsis which provides the capability to carry out full text searches on a large body of research literature.

Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene

BackgroundEukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.ResultsWe describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.ConclusionsThese results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants.

PiggyBac transposon-based gene delivery with cationic ultrasound contrast agents into SCID and CD-1 mice.

Efficient integration of a functional gene, along with suitable transcriptional regulatory elements, poses a fundamental difficulty for the application of successful gene therapy. Though inducible gene expression after somatic cell gene transfer has been achieved by employing viral vectors, issues associated with cargo capacity, host immune response, the risk of insertional mutagenesis, and the requirement for separate viruses are limiting factors. Several researchers have used transposon-based approaches for gene delivery, and the piggyBac transposon system has recently been shown to be effective in human cell lines and for the transgenesis in mice. The use of engineering helper-independent plasmids with the mouse codon-biased piggyBac transposon (mPB) gene in combination with cationic ultrasound contrast agents (UCAs) is investigated for a range of insonification parameters. The plasmid pmGENIE-3 deactivates the mPB gene after insertion of the transposon, eliminating potentially negative effects which may occur from the persistence of an active piggyBac gene post-transposition.

Search for conjugative transposon in rumen bacterium Prevotella bryantii B14

Only few plasmids and bacteriophages have been described to date in ruminal prevotella strains, therefore it appears plausible that the genetic exchange in these organisms must exploit other routes. Large conjugative transposons make possible the gene exchange process in bacteria from the genus Bacteroides, the phylogenetic relatives of ruminal prevotellas. The access to fully or partially finished genome sequences of Bacteroides and Prevotella representatives made possible the search for conserved regions within putative conjugative transposons. Multiple sequence alignment of known and putative conjugative transposon gene sequences of Bacteroides thetaiotaomicron, Prevotella intermedia, Bacteroides fragilis and Tanerella sp. was used to locate partially conserved regions within most preserved conjugative transposition genes, traG, and to construct appropriate degenerated oligonucleotide primers. These were used to amplify genome fragments from ruminal prevotella strains. Sequence analysis of the subcloned PCR products revealed the presence of a hypothetical gene in the genome of Prevotella bryantii B14, similar to the ORF BF2880 from B. fragilis YCH46, which is a part of a large conjugative transposon. Inverse PCRs were designed and performed to confirm the initial findings. A partial map of P. bryantii B14 putative conjugative transposon region was constructed, indicating an intergeneric horizontal gene transfer.

Gene Transfer Targeting Hepatocytes Using the PiggyBac Transposon System: An Approach towards Hemophilia A Correction and Long Term Expression of Factor VIII.

Abstract 3575Poster Board III-512Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBAC can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBAC transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008-15013, 2006). To date, there are no published reports of in vivo gene transfer to mammalian livers using the piggyBAC transposon system. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and the ability to excise itself precisely without leaving a footprint. We hypothesize that a piggyBAC transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PiggyBAC transposons were engineered to carry a hygromycin resistance gene (Hygro), a luciferase expression cassette (PB luciferase), or a human alpha1 antitrypsin reporter (hAAT). We evaluated co- transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB hygro vector, we demonstrated that the co- transposase generated higher transposition efficiency than an inactive mutant in hepatocytes. We then showed in vivo persistence following hydrodynamic tail-vein injection using firefly luciferase expression driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Luciferase expression measured via in vivo bioluminescence imaging persisted up to eight months in C57Bl/6 liver (duration of experiment). Following partial hepatectomies at 5 months post injection, expression was observed only in animals receiving PB luciferase transposon and an active transposase while expression in those treated with the inactive mutant dropped to background levels supporting that expression was from integrated transgene. We furthered these experiments by introducing PB hAAT via hydrodynamic tail-vein injection as before at either a low (12.5 micrograms each transposon and transposase) or high (50 micrograms each) dose. Serum hAAT levels were measured at 421ng/ml and 365ng/ml via ELISA at 3 months post-injection, respectively. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data represent one of the first studies to show persistent transgene expression in vivo from piggyBAC transposon gene transfer. Disclosures:No relevant conflicts of interest to declare.

Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants

Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans that are implicated in diverse developmental processes, including plant sexual reproduction. Male gametogenesis (pollen grain development) is fundamental to plant sexual reproduction. The role of two abundant, pollen-specific AGPs, AGP6, and AGP11, have been investigated here. The pollen specificity of these proteoglycans suggested that they are integral to pollen biogenesis and their strong sequence homology indicated a potential for overlapping function. Indeed, single gene transposon insertion knockouts for both AGPs showed no discernible phenotype. However, in plants homozygous for one of the insertions and heterozygous for the other, in homozygous double mutants, and in RNAi and amiRNA transgenic plants that were down-regulated for both genes, many pollen grains failed to develop normally, leading to their collapse. The microscopic observations of these aborted pollen grains showed a condensed cytoplasm, membrane blebbing and the presence of small lytic vacuoles. Later in development, the generative cells that arise from mitotic divisions were not seen to go into the second mitosis. Anther wall development, the establishment of the endothecium thickenings, the opening of the stomium, and the deposition of the pollen coat were all normal in the knockout and knockdown lines. Our data provide strong evidence that these two proteoglycans have overlapping and important functions in gametophytic pollen grain development.

New Frontiers in the Therapy of Tuberculosis: Fighting with the Global Menace

Tuberculosis has remained an enemy of humankind since its inception. It has affected all facets of human life and remained leading cause of mortality and morbidity despite of availability of effective chemotherapy and BCG vaccine in 21st century. This has exposed the frailties of the current drug armamentarium. No new drug is available acting through novel mechanism of action for last 40 years. This has culminated into resistant strain of TB, MDR-TB and XDR-TB. Concomitant occurrence of TB and HIV presents a lethal combination. The availability of the M. tuberculosis genome sequence and mycobacterial genetic tools, such as transposon mutagenesis, gene knockout and gene transfer, greatly facilitate target identification. This review provides a comprehensive literature compilation on the present research paradigm of anti-TB drug discovery including advances in the new structural classes analogs reported in last decade.

Microarray analysis of antimicrobial resistance genes inSalmonella entericafrom preharvest poultry environment

To detect antimicrobial resistance genes in Salmonella isolates from turkey flocks using the microarray technology. A 775 gene probe oligonucleotide microarray was used to detect antimicrobial resistance genes in 34 isolates. All tetracycline-resistant Salmonella harboured tet(A), tet(C) or tet(R), with the exception of one Salmonella serotype Heidelberg isolate. The sul1 gene was detected in 11 of 16 sulfisoxazole-resistant isolates. The aadA, aadA1, aadA2, strA or strB genes were found in aminoglycoside-resistant isolates of Salm. Heidelberg, Salmonella serotype Senftenberg and untypeable Salmonella. The prevalence of mobile genetic elements, such as class I integron and transposon genes, in drug-resistant Salmonella isolates suggested that these elements may contribute to the dissemination of antimicrobial resistance genes in the preharvest poultry environment. Hierarchical clustering analysis demonstrated a close relationship between drug-resistant phenotypes and the corresponding antimicrobial resistance gene profiles. Salmonella serotypes isolated from the poultry environment carry multiple genes that can render them resistant to several antimicrobials used in poultry and humans. Multiple antimicrobial resistance genes in environmental Salmonella isolates could be identified efficiently by microarray analysis. Hierarchical clustering analysis of the data was also found to be a useful tool for analysing emerging patterns of drug resistance.

Reprogramming the epigenome during germline and seed development

Gene silencing by DNA methylation and small RNAs is globally reconfigured during gametogenesis in Arabidopsis.

Heart-specific isoform of tropomyosin4 is essential for heartbeat in zebrafish embryos

Tropomyosin (Tpm) proteins, encoded by four Tpm genes (Tpm1-4), are associated with the stabilization of the F-actin filaments and play important roles in modulating muscle contraction. So far, little is known about Tpm4 function in embryonic heart development and its involvement in the cardiovascular diseases. In this study, we investigated functions of different isoforms of tpm4 in embryonic heartbeat in zebrafish. The transgenic zebrafish line, T2EGEZ8, was generated by insertion of a Tol2 transposon gene trap vector, and homozygous mutants (T2EGEZ8(m/m)) of this line showed failure of embryonic heartbeat without other detectable phenotypes. Observation by transmission electron microscopy revealed that the ventricular myocytes of mutant fish contained fewer, disorganized myofibrillar filaments. The transposon genome in T2EGEZ8 fish was found by thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and reverse transcription-polymerase chain reaction to have inserted into the ninth intron of the tpm4 locus, which resulted in production of Tpm4-GFP fusion proteins and loss of normal transcripts tpm4-tv1 and tpm4-tv2. Whole-mount in situ hybridization indicated that tpm4-tv1, encoding a peptide of 284 residues, is specifically expressed in the heart of zebrafish embryos, while tpm4-tv2, encoding a peptide of 248 residues, is mainly present in the vasculature but absent in the heart. Knockdown of tpm4-tv1 and tpm4-tv2 within wild-type embryos led to the failure of heartbeat, which could be rescued by coinjection with tpm4-tv1 mRNA but not with tpm4-tv2 mRNA. Tpm4-tv1 is a heart-specific isoform of Tpm4 and is essential for heartbeat in zebrafish embryos.

Epigenetic Inheritance and Reprogramming in Plants and Fission Yeast

Plants and fission yeast exhibit a wealth of epigenetic phenomena, including transposon regulation, heterochromatic silencing, and gene imprinting. They provide excellent model organisms to address the question of how epigenetic information is propagated to daughter cells. We have addressed the questions of establishment, maintenance, and inheritance of heterochromatic silencing using the fission yeast Schizosaccharomyces pombe and the plant Arabidopsis thaliana by using a variety of genetic and genomic approaches. We present here results showing the cell cycle dependence of RNA in fission yeast RNA interference (RNAi), which is required for proper transcriptional silencing of the centromeric heterochromatin, and that this process occurs during S phase, allowing for precise copying and reestablishment of heterochromatic histone modifications following DNA replication and cell division. We also show that in plants, cells in culture and male germ-line cells undergo massive epigenomic changes correlated with the appearance of a novel class of 21-nucleotide small interfering RNA (siRNA) from transcriptionally reactivated transposable elements (TEs) following loss of heterochromatic DNA and histone methylation. We propose a model for the role of deliberate TE reactivation in germ-line companion cells as part of a developmental mechanism for first revealing and then silencing TEs via small RNA, which may contribute to reprogramming during early development in plants and animals.

Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

BackgroundThus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB) transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy.ResultsTwo well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP) reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5) into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.ConclusionSB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins and thus confer resistance against viral infection by restricting viral entry. These studies have demonstrated for the first time the utility of the non-viral SB system in conferring stable resistance against HIV infection and paved the way for the use of this system for HIV gene therapy studies.

Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Sigma1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth.

Structure and conservation of a polyethylene glycol-degradative operon in sphingomonads

Sphingopyxis terrae, and Sphingopyxis macrogoltabida strains 103 and 203, can degrade polyethylene glycols (PEGs). They differ in the following respects: (i) different substrate specificities (chain length) of assimilable PEG, (ii) PEG-inducible or constitutive PEG-degradative proteins, and (iii) symbiotic or axenic degradation of PEG. S. terrae was able to incorporate PEG 6000, but strain 103 could not incorporate more than PEG 4000, suggesting that the difference in assimilable PEG chain length depends on the ability to take up substrate. PEG-degradative genes (pegB, C, D, A, E and R) from these strains were cloned. Their primary structures shared a high homology of more than 99 %. The peg genes encode a TonB-dependent receptor (pegB), a PEG-aldehyde dehydrogenase (pegC), a permease (pegD), a PEG dehydrogenase (pegA) and an acyl-CoA ligase (pegE), and in the opposite orientation, an AraC-type transcription regulator (pegR). The peg operon was flanked by two different sets of transposases. These three strains contained large plasmids and the operon was located in one of the large plasmids in S. terrae. The peg genes could be detected in other PEG-degrading sphingomonads. These results suggest that the peg genes have evolved in a plasmid-mediated manner. An insertion of a transposon gene (pegF) between pegD and pegA in strain 203 was found, which caused the constitutive expression of pegA in this strain.

Somatic transformation efficiencies and expression patterns using the JcDNV and piggyBac transposon gene vectors in insects

A somatic transformation gene vector that exploits the genomic integration properties of Junonia coenia lepidopteran densovirus (JcDNV) sequences in vivo has been developed. JcDNV somatic transformation vectors are derivatives of plasmids containing an interrupted genome of JcDNV that provide efficient, robust vectors that can be used to examine regulation of chromosomally integrated transgenes in insects. Microinjection of JcDNV plasmids into syncytial embryos of Drosophila melanogaster or the lepidopterans Plodia interpunctella, Ephestia kuehniella or Trichoplusia ni resulted in persistent transgene expression throughout development. Inclusion of transgenes with tissue-specific promoters resulted in expression patterns canonical with phenotypes of piggyBac germline transformants. Somatic transformation required the presence of the viral inverted terminal repeat in cis only and did not depend upon non-structural viral proteins.

Looking Farther Afield

Looking Farther Afield