title: Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens.
abstract: 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.
authors.affiliation: E.L. Ginzton Lab, Stanford University, Stanford, CA 94305-4085, USA. zappe@cmu.edu
authors.forename: Stefan
authors.initials: S
authors.lastname: Zappe
authors.forename: Matthew
authors.initials: M
authors.lastname: Fish
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Scott
authors.forename: Olav
authors.initials: O
authors.lastname: Solgaard
mesh_terms: D000818:Animals; D051186:Armadillo Domain Proteins / Q000096:biosynthesis / Q000235:genetics; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D046210:Microfluidic Analytical Techniques; D008845:Microinjections; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
chemical_list: C495305:ARM protein, Drosophila; D051186:Armadillo Domain Proteins; D029721:Drosophila Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D014157:Transcription Factors