Patterned delivery and expression of gene constructs into zebrafish embryos using microfabricated interfaces.
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.
Bansal
Tushar
T
Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI, USA. tbansal@umich.edu
Lenhart
Justin
J
Kim
Taesung
T
Duan
Cunming
C
Maharbiz
Michel M
MM
D000818:Animals; D004247:DNA / Q000235:genetics; D018274:Electroporation / Q000295:instrumentation*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004867:Equipment Design; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D005898:Glass / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D008839:Microelectrodes; D044085:Microfluidics / Q000295:instrumentation; D055616:Microtechnology; D008970:Molecular Weight; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity; D014966:Xanthenes / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D014966:Xanthenes; D049452:Green Fluorescent Proteins; C034657:Texas red; D004247:DNA; D019793:Fluorescein