DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Lan, Qing; Riddiford, Lynn M.

doi:10.1007/s11626-997-0111-5

Cited by 17 publications

(24 citation statements)

References 21 publications

(26 reference statements)

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“…To determine if a non-transposable DNA vector delivered to vitellogenic females could be used to over-express a heterogeneous gene in the next generation (F0), a β-galactosidase (β-gal) reporter gene construct driven by the 194 bp hsp70 promoter (pXH70ZT) [15] was used to monitor β-gal gene expression from DNA vectors in F0. The jetPEI/pXH70ZT complex was microinjected into the hemocoel in 10–15 vitellogenic females at 24 hours PBM via the thorax.…”

Section: Resultsmentioning

confidence: 99%

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

et al. 2011

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A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.

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Section: Resultsmentioning

confidence: 99%

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

et al. 2011

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“…Previous knowledge about the activity of the Drosophila hsp70 promoter outside Drosophila comes merely from transient expression of reporter genes using Manduca (Lan and Riddiford 1997), Bombyx (Kimura et al 1999) or mosquito (Zhao and Eggleston 1999) cell lines. Although cell lines are extremely useful, they do not fully reflect the regulation of a gene in a live animal.…”

Section: Transgenic Expression Is Rapidly Induced In Vivomentioning

confidence: 99%

“…Lepidopteran larvae have been known to respond to warming by expressing heat shock proteins (Fittinghoff and Riddiford 1990) and studies with two lepidopteran cell lines (Lan and Riddiford 1997;Kimura et al 1999) encouraged the idea that hsp70 promoter might also be active in vivo.…”

Section: Introductionmentioning

confidence: 99%

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Uhlířová

Asahina

Riddiford

et al. 2002

Development Genes and Evolution

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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.

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“…Drosophila heat shock protein70 (HSP70) promoter (Thummel and Pirrotta, 1992), which had been used successfully in conditional gene expression in different insect cell lines (Kimura et al, 1999;Lan and Riddiford, 1997;Zhao and Eggleston, 1999) and intact animals (Uhlirova et al, 2002) including Lepidoptera (Fittinghoff and Riddiford, 1990;Uhlirova et al, 2002), was selected to drive a heat shock inducible transcription of the double-stranded RNA.…”

Section: Introductionmentioning

confidence: 99%