Microinjection in C. elegans by direct penetration of elastomeric membranes

Abstract

The nematode worm C. elegans is widely used in basic and translational research. The creation of transgenic strains by injecting DNA constructs into the worm's gonad is an essential step in many C. elegans research projects. This paper describes the fabrication and use of a minimalist microfluidic chip for performing microinjections. The worm is immobilized in a tight-fitting microchannel, one sidewall of which is a thin elastomeric membrane through which the injection pipet penetrates to reach the worm. The pipet is neither broken nor clogged by passing through the membrane, and the membrane reseals when the pipet is withdrawn. Rates of survival and transgenesis are similar to those in the conventional method. Novice users found injections using the device easier to learn than the conventional method. The principle of direct penetration of elastomeric membranes is adaptable to microinjections in a wide range of organisms including cells, embryos, and other small animal models. It could, therefore, lead to a new generation of microinjection systems for basic, translational, and industrial applications.

FIG. 1.

Layout and assembly of the injection chip. (a) Top view. Colors: light yellow, PDMS; blue, fluid-filled features; gray, glass coverslip. (b) Area of detail indicated in (a). Upper panel, top view; lower panel, side view of a-a’ transect. (c) Assembly of chip, coverslip, and frame.

FIG. 2.

(a) Photograph of the assembled device. (b) Top view of the injection chip filled with blue ink to visualize the vestibule, injection channel, and post-injection reservoir. (c) Close-up view of the injection channel, septum, and cut-out. The scale bar is 100 μm.

FIG. 3.

Injection chip mold. Top and bottom plates are joined by corner screws (1). The nose feature is positioned by turning a screw (2). The nose is locked into position by screw (3).

FIG. 4.

Optimal injection-pipet shape. (a) Photomicrograph of an injection pipet after use. The ovoid object is a droplet of oil that remained on the pipet. (b) Model of the tip shown in (a). The diameter of the pipet at points a, b, and c is 5.4, 1.8, and ∼0.7 μm, respectively. Proximal and distal are defined relative to the position of worm's vulva, which is located on the ventral midline.

FIG. 5.

Graphical representation of survival rates and transformation rates given in Table I. Rates are expressed as probabilities. Error bars are 95% confidence intervals.

FIG. 6.

Processing rates for each stage of the Poker Chip injection procedure and overall process time for Poker Chip and conventional injections. Data are in units of minutes per worm. Numbers associated with bars are mean values. Statistical comparisons (t-test), Conventional vs Poker Chip, Total time: d.f. combined = 5.88, t = 4.07, p = 0.0069. Error bars are 95% confidence interval.