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At least 901 records · Page 50Linked to original sources
PubMed2003

Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells.

Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E. coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

Animals↗
PubMed2002

Field evaluation of three plant-based insect repellents against malaria vectors in Vaca Diez Province, the Bolivian Amazon.

The efficacy of repellents against Anopheles darlingi, the main malaria vector in Bolivia, was evaluated. This mosquito has a peak in biting activity early in the evening. Three natural repellents (1 eucalyptus based, 1 neem based, and 1 containing several repellent essential oils) were tested in comparison with 15% deet in human landing catches in Bolivia. The eucalyptus-based repellent containing 30% p-menthane-diol applied at a dose similar to those used in practice gave 96.89% protection for 4 h. Deet gave 84.81% protection. The other 2 products did not provide significant protection from mosquito bites.

Animals↗
PubMed1997

Expression of neuroparsin cDNA in insect cells using baculovirus vectors.

The cDNA encoding neuroparsin A, a polytropic neurohormone of the locust, Locusta migratoria, was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that transcription was under control of the p10 promoter. A polypeptide having the same charge and the same apparent molecular weight as the authentic neuroparsin A and that was reactive against neuroparsin immune serum was produced in recombinant virus-infected lepidopteran cell lines but not in control virus-infected cells. The baculovirus-expressed polypeptide was purified by two steps of liquid chromatography (anion exchange and reversed phase) which were previously used to purify the natural neuroparsin. The purified baculovirus-expressed polypeptide enhanced fluid reabsorption of everted rectum preparations, as did the natural neuroparsin. Thus, this gene expression system produced a polypeptide identical to authentic neuroparsin.

Animals↗
PubMed1996

Baculovirus vectors for expression in insect cells.

Recombinant baculoviruses now represent a mature technology in which vector development, particularly for the control of expression level, has reached a plateau. However, other aspects of expression, such as the production of multiple proteins, improved product purification or maximizing protein processing, remain areas for novel vector and host cell development. This year has seen these topics come to the fore in descriptions of new expression systems.

Animals↗
PubMed2004

RNAi-mediated inhibition of gene function in the follicle cell layer of the Drosophila ovary.

RNA-mediated interference (RNAi) has been reported to be an effective reverse genetic approach for studying gene function in various organisms. To assess RNAi as a means of examining genes expressed in ovarian follicle cells for their involvement in embryonic dorsal-ventral patterning, we tested the ability of transgenically expressed double-stranded RNA (dsRNA) directed against the dorsal group gene windbeutel to generate phenotypic effects in the progeny of expressing females. We observed that expression in follicle cells under the control of Gal4 transcribed from the strong and widely expressed alphaTub84B or Actin5C promoters led to efficient dorsalization of progeny embryos. Surprisingly, a variety of strongly expressed follicle cell-specific Gal4 enhancer trap lines failed to elicit an RNAi phenotype in combination with the windbeutel-specific dsRNA. These results stress the importance of careful choice of expression system and of conditions for use in transgenic RNAi-mediated studies of gene function.

Animals↗
PubMed2004

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea.

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.

Amino Acid Sequence↗
PubMed2003

Integrated control of vector-borne diseases of livestock--pyrethroids: panacea or poison?

Tick- and tsetse-borne diseases cost Africa approximately US$4-5 billion per year in livestock production-associated losses. The use of pyrethroid-treated cattle to control ticks and tsetse promises to be an increasingly important tool to counter this loss. However, uncontrolled use of this technology might lead to environmental damage, acaricide resistance in tick populations and a possible exacerbation of tick-borne diseases. Recent research to identify, quantify and to develop strategies to avoid these effects are highlighted.

Animals↗