Injecting Gryllus bimaculatus Eggs

Abstract

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Figure 1:. Egg injection protocol overview.

A) A typical set up that can be used for egg injections. B) Eggs are separated from sand via a sieve. C) Eggs are held for injection in small wells made by placing a mold into warm, liquid agarose. D) Fluorescence in the needle and in five injected eggs can be seen through the fluorescent dissecting microscope. E) An uninjected egg. The anterior end (slightly pointed; left) and posterior end (more blunted; right) are distinguishable from each other. The region most suitable for injection, near the posterior end, is shown with the bracket. F) An egg two days after injection. The injection site is visible as a slight discoloration, and a small amount of expelled yolk is evident (arrow). Scale bars D: 1 mm; E and F: 0.5 mm.

Figure 2:. Needles are beveled using a micro grinder or beveller.

A) An example of a micro grinder with a circular, spinning grinding surface moistened by dripping RO water (water drips from the syringe shown in the top of the image). B) Before beveling, pulled needles are long and narrow. C) After beveling to 20°, a sharp injection needle is created. Note the 10 μm diameter of the inner lumen. (Red scale bars are 10 μm). D) Pulled, beveled needles can be stored in a Petri dish with a lid and held in place using dental wax.

Figure 3:. Injections can decrease rate of survival.

A) Dead and dying eggs are obvious upon visual inspection. B) Over time, material will migrate to one side of the egg. C) Comparison of average survival rates four to six days after egg laying for a variety of conditions, including: uninjected eggs (n=6 experiments); eggs punctured with a 9 μm needle but uninjected (n=2 experiments); eggs injected with buffer and dye (n=13 experiments); eggs injected with buffer, dye, and plasmid vector (n=2 experiments); eggs injected with buffer, dye, and DMSO (n=11 experiments); and eggs injected with buffer, dye, and control dsRNA (n=22 experiments). The type of control dsRNA used included dsRNA against eGFP or dsRed. The numbers at the base of each bar note the total number of surviving eggs/total eggs for for each condition. Error bars represent standard error of the mean. Scale bar for A and B shown in B = 1 mm.

Figure 4:. Assessing injection results.

(A-B) Injection of Enhancer of Zeste (E(z)) dsRNA alters the normal expression of Ubx mRNA (wild type shown in A), and leads to the transformation of antennae (AN) and mouthparts (Mx, Lb) into leg-like appendages (B). (C-D) Injection of early eggs with the transposable element piggyBac and histone2B-eGFP produces transgenic embryos expressing GFP in all nuclei. Location of nuclei can be observed eight h after egg laying (C) and 20 h after egg laying (D). Abbreviations: An: Antenna; Mx: maxilla; Lb: labium; T1–3: thoracic legs 1 to 3; RNAi: RNA interference; Gb: G. bimaculatus; Act: actin; H2B; Histone H2B; GFP: green fluorescent protein. Scale bars: 200 μm for A and B; 500 μm in E and F. These experimental results were originally described elsewhere^, .