Regulation of <i>Junonia coenia</i> densovirus P9 promoter expression

Shirk, Paul D.; Bossin, Hervé; Furlong, Richard B.; Gillett, J. L.

doi:10.1111/j.1365-2583.2007.00759.x

Cited by 12 publications

(14 citation statements)

References 35 publications

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“…Therefore, inclusion of cultured honeybee cells [64] may provide a more attractive and complete feeding medium. Because the cultured insect cells can be genetically transformed [65, 66], supplementation of the feeding medium can provide an additional means of introducing various constructs that will produce materials that can be tested for control of the mites.…”

Section: Discussionmentioning

confidence: 99%

A feeding protocol for delivery of agents to assess development in Varroa mites

2017

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A novel feeding protocol for delivery of bio-active agents to Varroa mites was developed by providing mites with honey bee larva hemolymph supplemented with cultured insect cells and selected materials delivered on a fibrous cotton substrate. Mites were starved, fed on treated hemolymph to deliver selected agents and then returned to bee larvae. Transcript levels of two reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as well as for nine selected genes involved in reproductive processes showed that the starvation and feeding protocol periods did not pose a high level of stress to the mites as transcript levels remained comparable between phoretic mites and those completing the protocol. The feeding protocol was used to deliver molecules such as hormone analogs or plasmids. Mites fed with Tebufenozide, an ecdysone analog, had higher transcript levels of shade than untreated or solvent treated mites. In order to extend this feeding protocol, cultured insect cells were incorporated to a final ratio of 1 part cells and 2 parts hemolymph. Although supplementation with Bombyx mori Bm5 cells increased the amount of hemolymph consumed per mite, there was a significant decrease in the percentage of mites that fed and survived. On the other hand, Drosophila melanogaster S2 cells reduced significantly the percentage of mites that fed and survived as well as the amount of hemolymph consumed. The feeding protocol provides a dynamic platform with which to challenge the Varroa mite to establish efficacy of control agents for this devastating honey bee pest.

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Section: Discussionmentioning

confidence: 99%

A feeding protocol for delivery of agents to assess development in Varroa mites

2017

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“…Since the ClaI cutting site, located in the pBR322 backbone, is used to generate every linear molecule, this effect is probably due to the AflII cutting site which was brought along with the gfp gene during the construction of plasmids. Restriction with AflII deleted the polyadenylation signals (poly-A) which belong to both ORF1 and ORF2/ORF3 in the ambisense organization of JcDV (Dumas et al 1992;Shirk et al 2007) and are present in all three constructs. This result is in agreement with the pivotal role of Polycomb Response Elements in this region, as described by Shirk et al (Shirk et al 2007).…”

Section: Jcdv-based Linear Vectors Drive Stable Expression Of Gfp Inmentioning

confidence: 99%

Peer Review #1 of "Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells (v0.1)"

2018

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Background. The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. Methods. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of GFP and DNA was analysed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. Results. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and nonviral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Conclusions. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Last, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

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“…In addition to transformation of insect cell lines, the JcDNV somatic transformation vectors have been used effectively to somatically transform various dipteran and lepidopteran species by microinjecting syncytial embryos (Royer et al, 2001;Bossin et al, 2007). Microinjection of JcDNV vectors led to the highly efficient recovery of somatic transformants where promoter specific expression was maintained from the larval through the adult stage (Royer et al, 2001;Bossin et al, 2007;Shirk et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

Shirk

Furlong

2016

Plasmid

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A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.

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Regulation of Junonia coenia densovirus P9 promoter expression

Cited by 12 publications

References 35 publications

A feeding protocol for delivery of agents to assess development in Varroa mites

A feeding protocol for delivery of agents to assess development in Varroa mites

Peer Review #1 of "Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells (v0.1)"

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

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