Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis

Abstract

We describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro-translated mRNAs or gene-specific oligonucleotide morpholinos (MOs), respectively. Our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. Embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. A micromanipulator is used to guide the needle to impale individual embryos. Injection takes ∼1.5 h, and an experienced researcher can inject ∼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in ∼1 week.

Figure 1

Microinjection. (a) Image of the injection apparatus. Shown is a typical setup for a right-handed injector. Injections are carried out using a standard stereo-dissecting microscope outfitted with a forced-air micropipette injection needle mount (1). A coarse (2) and fine (3) micromanipulator allow for mechanical control of the needle during the injection process. A single oblique-angled light source (4) allows the researcher to manipulate the dish and use their hand to both manipulate the injection dish and modulate white light. Fluorescence is excited by an external light source (5). A picospritzer (6) is used to control the flow of air to the needle, and an injection foot pedal (7) allows the injector to pulse air for injection while leaving hands free to hold the injection dish and the micromanipulator. (b) Example of injection dish with two rows of embryos positioned and ready for injection. Inset in b shows a 1-cm ruler with scale of spacing for scratches on the bottom of an injection dish. (c) Three injection needles with tips indicated by white arrowheads. Ruler scale is 1 mm per line. All three needles are sufficient for injection. The one on the left is stout and cannot be broken back as much as the other two if there is clogging. The one on the right has a very fine tip and can be flimsy, making injections for beginners more difficult. The middle needle combines the properties of both stoutness and a fine flexible tip.

Figure 2

Expression of Venus protein in Nematostella injected with NvashA:venus mRNA. (a–d) Z-projection (8 μm) of cleavage stage embryo 3 h after injection with NvashA:venus mRNA. Venus expression can be clearly observed in c,d. Nuclear localization of Venus is observed by using Hoechst as a counterstain to label DNA (b–d). Nuclear localization is expected because the venus coding sequence is fused in-frame with the NvashA transcription factor coding sequence, which contains a nuclear localization signal. (e–l) Low magnification views of unsorted embryos grown at 17 °C for 24 h after injection. Embryos were co-injected with dextran and NvashA:venus mRNA (e–h) or dextran alone (i–l). Nearly all embryos co-injected with the dextran and mRNA show strong fluorescence from the Venus protein (g,h), whereas control animals do not show green fluorescence (k,l). DIC, differential interference contrast. Scale bars (a–d), 100 μm; (e–l), 500 μm.

Figure 3

Assaying MO efficiency. (a) Sequence information for Nvtcf (wild-type), Nvtcf:Venus (only ORF), Nvtcf5′:Venus (containing part of the 5′ UTR) and the target sequence for MoTcf_trans. (b–e) Overexpression of Nvtcf:Venus (b) or Nvtcf5′:Venus (d) alone or in presence of MoTcf_trans (c,e) showing that MoTcf has no effect on Nvtcf:Venus translation, whereas MoTcf inhibits translation of Nvtcf5′:Venus. The green dots correspond to the nuclear localization of the injected product. (f) Schematic of the genomic organization of NvetsA1. The approximate positions of the exons are indicated by blue boxes. F1 and R2 indicate the positions of the primers used to assay the efficiency of the splice-blocking MO. In uninjected controls, only the amplicons corresponding to spliced versions of the transcript (440 bp) are detected, whereas in MoEtsA1-injected embryos the large majority of the PCR product corresponds to an unspliced version of the transcript (800 bp). The low level of the 440-bp fragment compared with the 800-bp fragment in injected embryos demonstrates the efficiency of the used MO. Scale bar in b–d, 100 μm.

Figure 4

Development after injection. (a) Survival curve of uninjected control (blue; n = 190), 500 nM control MO injected (red; n = 159), 200 ng μl ⁻¹ dextran (green; n = 221) and 300 ng μl ⁻¹ gfp mRNA (purple; n = 183). Animals were developed at 22 °C. (b) Ratio of surviving animals that had developed to the four-tentacle juvenile polyp stage by 144 h after fertilization. Control n = 168, control MO n = 142, dextran n = 185 and gfp mRNA n = 140.