Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

Thomas, Jean-luc

doi:10.1673/031.003.1701

Cited by 12 publications

(6 citation statements)

References 54 publications

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“…Initially developed for transfecting in vitro cultured cells [19], the technique was adapted to ex-vivo , in-situ and in vivo DNA transfection of tissue or whole organisms [20]. The principle application of electroporation is focused on vertebrate tissues and organisms with no previous work having been done using ticks.…”

Section: Introductionmentioning

confidence: 99%

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

2010

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BackgroundTicks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation.ResultsThe genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs.ConclusionsOur study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.

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Section: Introductionmentioning

confidence: 99%

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

2010

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“…Thomas (2003) also attempted similar experiments using Bombyx mon embryos and dissected larval wings and ovaries. He found that 2 out of 16 wings expressed some number of cells expressing Lac Z using 5 pulses of 250 V, 50 ms pulse duration, and 1 s between pulses.…”

Section: Resultsmentioning

confidence: 99%

“…The use of electroporation as a genetic transformation method of broad application across species has proliferated in recent years (Moto et al 1999; Zhang et al 1999; Saito and Nakatsuji 2001; Haas et al 2002; Tawk et al 2002; Thomas 2003; Sano et al 2003; Kunieda and Kubo 2004). Electroporation introduces foreign DNA into cells by temporarily opening pores for the DNA to enter the cell, enabling the study of regulation and function of these genes and proteins (Chang and Reese 1990).…”

Section: Introductionmentioning

confidence: 99%

In Vivo Electroporation of DNA into the Wing Epidermis of the Butterfly,Bicyclus anynana

Golden

Sagi

Markwarth

et al. 2007

Journal of Insect Science

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The direct transfer of genes into differentiated insect tissues is a useful method of determining gene function because it circumvents the need to perform germ line transformations and of having information on tissue-specific gene promoters. Here in vivo gene delivery is achieved through electroporation of a reporter gene into the pupal forewing of the butterfly Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae). Plasmids containing the coding sequence for enhanced green fluorescent protein (EGFP), driven by the Drosophila heat-shock promoter hsp70, were successfully expressed in epidermal cells after plasmid injection followed by electroporation and heat shock. EGFP expression was restricted to the vicinity of the injection and electroporation site, but the number of transformed cells varied from a few to over 5000 cells. Electroporation parameters were optimized in order to maximize the number of transformed cells while minimizing the extent of damage to the adult wing. While certain electrical parameters were well tolerated by the wing tissue, the physical damage caused by the insertion of the tungsten electrodes led to frequent disruptions of the adult wing pattern around the puncture sites. While this technique can be useful to test the correct expression of marker genes (such as EGFP) in newly build plasmids immediately following their injection, its potential use in testing the function of candidate genes in wing pattern formation is limited.

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“…Injection of early-stage embryos allows DNA, RNA and protein-based technologies to be incorporated into embryonic cells that will go on to form the germ line, resulting in some cases in heritable changes in the genome. Alternatives to micro-injection of early-stage embryos such as biolistics and electroporation have been reported and while somatic delivery using these methods could be confirmed, germ-line modifications have not been reported in vector insects following these modes of delivery [8,9].…”

Section: Technologies For Moving Genetic Technologies Into Genomesmentioning

confidence: 99%

Genetic technologies for disease vectors

Criscione

O’Brochta

Reid

2015

Current Opinion in Insect Science

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Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

Cited by 12 publications

References 54 publications

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

In Vivo Electroporation of DNA into the Wing Epidermis of the Butterfly,Bicyclus anynana

Genetic technologies for disease vectors

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