Microtechnology for Spatial and Temporal Control of Gene Expression in Developing Embryos and Cells.

Abstract

Much of biology in multicellular development depends upon the exchange of messages between cells: by exchanging chemical or mechanical information, cells influence the state of other cells near them and act in a coordinated fashion. Many tissue patterning events such as migration, proliferation and differentiation that occur in vivo, occur in relation to changes in local microenvironment of a cell. The ability to modify two dimensional specially-shaped areas of the local microenvironment with controlled delivery of genes and proteins in patterned shapes allows more biologically-relevant manipulations of a complete system’s development. Such ‘hacking’ tools are limited at this time and our technology provides a biological tool to study more of such developmental mechanisms. The work presented in this thesis focuses on the design, fabrication and application of microfabricated interfaces for the patterned delivery of foreign molecules and gene constructs into developing embryos and cells. Low voltage microfluidic valves and pumps were designed to deliver multiple compounds in pixel style resolution into growing embryos. Other systems were 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. The successful delivery of two-dimensional patterns of trypan blue (normal dye), texas red (fluorescent dye), pCS2eGFP DNA and GFP-mRNA in both chorionated and dechorionated embryos was also demonstrated. Both DNA and mRNA were expressed in the desired patterns subsequent to delivery. Briefly, 10 µm wide platinum electrodes were microfabricated into desired patterns and passivated with silicone. Square pulses of 10-20 V (0.20-0.40 kV/cm), 50-100 ms pulse 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 rate of 91.3% and 89%, and a delivery rate of 38% and 50% for GFP- DNA and GFP-mRNA respectively. We believe that these simple techniques offer the unique advantages of introducing foreign compounds at local sites and in specific patterns unlike any other microsystem techniques and provide new tools to aid advanced studies in cellular development and morphogenesis.