An electroporation microchip system for the transfection of zebrafish embryos using quantum dots and GFP genes for evaluation

Huang, Keng‐Shiang; Lin, Yu-Cheng; Su, Kai-Chun; Chen, Hung-Yi

doi:10.1007/s10544-007-9087-x

Cited by 28 publications

(21 citation statements)

References 41 publications

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“…Although the advantages of miniaturisation have been widely recognised when dealing with single cells (Boukany et al, 2011;Fox et al, 2006;Wang et al, 2010;Wang and Lee, 2013), microfabrication techniques have only rarely been adopted for the electroporation of developing organisms (Bansal et al, 2009;Huang et al, 2007). To our knowledge this work validates for the first time the superiority of dielectric guide based designs when targeting small cell populations in embryos.…”

Section: Discussionsupporting

confidence: 52%

“…Xenopus (Chernet and Levin, 2012;Falk et al, 2007), chick (Nakamura et al, 2004;Voiculescu et al, 2008), zebrafish (Bansal et al, 2009;Huang et al, 2007), insects (Ando and Fujiwara, 2013); organ explants, e.g. brain (Barker et al, 2009;del Rio and Soriano, 2010), retina (Matsuda and Cepko, 2004), cochlea (Driver and Kelley, 2010), gut (Abud et al, 2008), gonads (Gao et al, 2011); or in vitro reconstituted tissues, e.g.…”

Section: Discussionmentioning

confidence: 99%

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A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

et al. 2014

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The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

et al. 2014

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“…However, the microscale channels fail to easily handle the multicellular organism of large size, such as zebrafish embryos, which might obscure their promise of application in compound-related toxicity and teratogenicity assay. Recently, there has been a trend for zebrafish-related study turning to lab-on-a-chip, such as high-efficient embryonic microinjection, 25 high-throughput embryonic droplets generation, 26 and digital transportation of droplet encapsulated embryo, 27 ranging from dynamic manipulation to static stimulation by electroporation 28 and microfluids. 29 However, the microscale droplet is ill suited for long-time culture of embryos, particularly in small-size segments, 26 and the autocrine or paracrine may affect their normal development.…”

mentioning

confidence: 99%

An integrated microfluidic array system for evaluating toxicity and teratogenicity of drugs on embryonic zebrafish developmental dynamics

et al. 2011

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Seeking potential toxic and side effects for clinically available drugs is considerably beneficial in pharmaceutical safety evaluation. In this article, the authors developed an integrated microfluidic array system for phenotype-based evaluation of toxic and teratogenic potentials of clinical drugs by using zebrafish ͑Danio rerio͒ embryos as organism models. The microfluidic chip consists of a concentration gradient generator from upstream and an array of open embryonic culture structures by offering continuous stimulation in gradients and providing guiding, cultivation and exposure to the embryos, respectively. The open culture reservoirs are amenable to long-term embryonic culturing. Gradient test substances were delivered in a continuous or a developmental stage-specific manner, to induce embryos to generate dynamic developmental toxicity and teratogenicity. Developmental toxicity of doxorubicin on zebrafish eggs were quantitatively assessed via heart rate, and teratological effects were characterized by pericardial impairment, tail fin, notochord, and SV-BA distance /body length. By scoring the teratogenic severity, we precisely evaluated the time-and dose-dependent damage on the chemical-exposed embryos. The simple and easily operated method presented herein demonstrates that zebrafish embryo-based pharmaceutic assessment could be performed using microfluidic systems and holds a great potential in high-throughput screening for new compounds at single animal resolution.

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“…) in suspension are introduced between two opposite electrodes. A high electric field, typically from 1 to 10 kV/cm, is applied to the electrodes to deliver the molecules into the cells (Huang et al 2007;Rambabu et al 2005;Lurquin 1997). Electroporation is a well-characterized method for molecule delivery into embryos of many species (Lurquin 1997;Swartz et al 2001), although microinjection is more common for zebrafish embryos.…”

Section: Device Theorymentioning

confidence: 99%

“…Various groups are starting to build microsystem platforms to introduce compounds into single cells and developing embryos (Khine et al 2005;Zappe et al 2006). Huang et al (2007) introduced the first microfabricated device to electroporate zebrafish embryos. Huang's technology reproduced the capabilities of conventional electroporators in chorionated embryos but with limited resolution.…”

Section: Introductionmentioning

confidence: 99%

Patterned delivery and expression of gene constructs into zebrafish embryos using microfabricated interfaces

Bansal

Lenhart

Kim

et al. 2009

Biomed Microdevices

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We demonstrate a method which uses simple microfabrication and microfluidics to produce custom, shaped electroporators for the patterned delivery of foreign molecules into developing embryos. We show how these electroporators can be used to 'draw' two-dimensional patterns of tracer molecules, DNA and mRNA into the yolk and cells of zebrafish embryos (Danio rerio) at different stages of development. We demonstrate the successful delivery of patterns of Trypan Blue (normal dye), Texas Red (fluorescent dye), GFP-expressing DNA plasmids and GFP expressing mRNA constructs into both chorionated and dechorionated embryos. Both DNA and mRNA were expressed in the desired patterns subsequent to delivery. Square pulses of 10-20 V (0.20-0.40 kV/cm), 50-100 ms width were sufficient to create transient pores and introduce compounds from the late blastula period (3 hpf) to early pharyngula period (24 hpf) embryos. Using 24 hpf dechorionated embryos, we achieved a high survival of 91.3% and 89%, and a delivery efficiency of 38% and 50% for GFP-DNA and GFP-mRNA respectively. Lastly, we demonstrate the simultaneous delivery of different compounds into the developing embryo.

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An electroporation microchip system for the transfection of zebrafish embryos using quantum dots and GFP genes for evaluation

Cited by 28 publications

References 41 publications

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

An integrated microfluidic array system for evaluating toxicity and teratogenicity of drugs on embryonic zebrafish developmental dynamics

Patterned delivery and expression of gene constructs into zebrafish embryos using microfabricated interfaces

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