Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens

E.L. Ginzton Lab, Stanford University, Stanford, CA 94305-4085, USA.
Lab on a Chip (Impact Factor: 6.12). 09/2006; 6(8):1012-9. DOI: 10.1039/b600238b
Source: PubMed


We have developed an automated system based on microelectromechanical systems (MEMS) injectors for reliable mass-injection of Drosophila embryos. Targeted applications are high-throughput RNA interference (RNAi) screens. Our injection needles are made of silicon nitride. The liquid to be injected is stored in an integrated 500 nl reservoir, and an externally applied air pressure pulse precisely controls the injected volume. A steady-state water flow rate per applied pressure of 1.2 nl s(-1) bar(-1) was measured for a needle with channel width, height and length of 6.1 microm, 2.3 microm and 350 microm, respectively. A typical volume of 60 pl per embryo can be reliably and rapidly delivered within tens of milliseconds. Theoretical predictions of flow rates match measured values within +/-10%. Embryos are attached to a glass slide surface and covered with oil. Packages with the injector chip and the embryo slide are mounted on motorized xyz-stages. Two cameras allow the user to quickly align the needle tip to alignment marks on the glass slide. Our system then automatically screens the glass slide for embryos and reliably detects and injects more than 98% of all embryos. Survival rates after deionized (DI) water injection of 80% and higher were achieved. A first RNAi experiment was successfully performed with double-stranded RNA (dsRNA) corresponding to the segment polarity gene armadillo at a concentration of 0.01 microM. Almost 80% of the injected embryos expressed an expected strong loss-of-function phenotype. Our system can replace current manual injection technologies and will support systematic identification of Drosophila gene functions.

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    • "Complementary efforts have sought to use MEMS fabrication techniques to create devices that improve injection reproducibility [6], or facilitate cell capture in ordered arrays for rapid identification and alignment [3] [4] [5] [7]. However, utility for ultrahigh throughput (UHT) microinjection continues to be limited by reliance upon serialized injection methodologies. "
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    ABSTRACT: We describe a massively-parallelized, MEMS-based device concept for passively delivering exogeneous molecules into living cells via mechanical membrane penetration, i.e., mechanoporation. Details regarding device design and fabrication are discussed, as are results from preliminary live cell studies focused on device validation at the proof-of-concept level. These efforts represent key steps towards our long-term goal of developing instrumentation capable of ultrahigh throughput (UHT) cellular manipulation via active microinjection.
    Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 08/2012; 2012:594-7. DOI:10.1109/EMBC.2012.6346001
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    • "The major limitation for assaying small molecules in the fly embryo is the impermeability of the eggshell. Microinjection methods, despite recent advancements (Zappe et al. 2006), continue to be labor intensive and require individual manipulation of each embryo in a dedicated facility. The ultimate barrier to delivery of small molecules to the embryo is the waxy layer that encompasses the vitelline membrane of the eggshell. "
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    ABSTRACT: Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of d-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995Da. We find that the embryo undergoes an age-dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP-reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high-throughput format.
    Insect biochemistry and molecular biology 11/2010; 40(11):792-804. DOI:10.1016/j.ibmb.2010.07.007 · 3.45 Impact Factor
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    • "However, switching from one embryo to another was conducted manually . A semi-automated high-throughput Drosophila embryo injection system was reported recently, where a single surface micromachined microelectromechanical systems (MEMS) needle was used as an injector [22]. One drawback of this system is that manual alignment of the injector and glass slide results in alignment errors that would greatly influence the injection performance. "
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    ABSTRACT: The ability of efficiently delivering soluable/insoluable drug compounds or biomolecules into individual biological cells and quantifying their cellular responses is important for genetics, proteomics, and drug discovery. This paper presents a fully automated system for zebrafish embryo injection, which overcomes the problems inherent in manual injection, such as human fatigue and large variations in success rates due to poor reproducibility. Based on ldquolooking-then-movingrdquo control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute. Besides a high injection speed that compares favorably with that of a highly proficient injection technician, a vacuum-based embryo holding device enables fast immobilization of a large number of zebrafish embryos, shortening the embryo patterning process from minutes to seconds. The recognition of embryo structures from image processing identifies a desired destination inside the embryo for material deposition, together with precise motion control resulting in a success rate of 100%. Carefully tuning suction pressure levels as well as injection and retraction speeds produced a high survival rate of 98%. The quantitative performance evaluation of the automated system was based on the continuous injection of 250 zebrafish embryos. The technologies can be extended to other biological injection applications such as the injection of mouse embryos, Drosophila embryos, and C. elegans to enable high-throughput biological and pharmaceutical research.
    IEEE Transactions on Automation Science and Engineering 05/2009; 6(2-6):209 - 219. DOI:10.1109/TASE.2008.917136 · 2.43 Impact Factor
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