Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

The overall goal of this procedure is to introduce mRNA, encoding a fluorescent protein into bronchos stoma lancia latam oocytes to allow in vivo fluorescent imaging of developing embryos. This is accomplished by first inducing spawning of ripe amfi ais, males and females by a temperature shock of 24 hours. Following the temperature shock, females and males are placed into individual spawning cups, allowing the collection of respectively oocytes and sperm.

Once the cytes are lined up in a polylysine coated dish, especially prepared microinjection needle is used to inject solution inside the core of the cyte. The final step is the fertilization of the cyte with one to five drops of sperm. Ultimately two photon laser scanning.

Microscopy is used to show the ubiquitous expression of the fluorescent proteins produced from the micro injected mRNA. Visual demonstration of this method is critical as microinjection steps are difficult to learn because the required technical skills that are impossible to detail and cover in print. To begin, prepare the polylysine coated dishes used to immobilize the cytes during the injection.

For each 35 millimeter cell culture Petri dish, cover the bottom with polylysine solution and incubate at room temperature for five minutes. Next, transfer the polylysine solution into another dish and again, incubate at room temperature for five minutes. Discard the solution and let the Petri dishes dry upside down at room temperature for two hours.

When dry store the coated dishes wrapped in a plastic wrap at four degrees Celsius for up to one week. To prepare the aros coated dishes used to culture injected embryos, make artificial seawater and reverse osmosis water. Next, dissolve aros to a 1%concentration in filtered a SW swiftly poured the warm aros solution from one Petri dish into another to ensure a very thin aros coating of the dish.

Wrap the coated dishes in Saran wrap and store at four degrees Celsius for up to one week. First, prepare the injection mix in RNAs and DNA free water phenol. Red colors the solution and allows for the identification of successfully injected embryos.

Next centrifuge the mix to pellet the crystals and keep on ice until ready to use. When ready, load a 10 microliter pipette with 0.5 microliters of the injection mix, avoiding the bottom of the tube where the crystals have been pelleted. Backfill the injection needle by pipetting the 0.5 microliter drop of injection.

Mix into the large opening of the needle. Prepare two needles in case one breaks during the injection. Store the needles in a jar with water at the bottom to prevent evaporation of the injection.

Mix and place at four degrees Celsius. Let the injection mix slowly travel to the tip of the injection needle for at least one hour to minimize the creation of bubbles. To begin oocyte and sperm collection, males and females are shocked in a SW for 24 hours and then transferred into individual cups one to two hours before spawning.

Upon spawning, immediately collect sperm and cytes. Take care not to startle the adults during collection. Additional movement can dissipate and hence dilute both sperm and cytes after collection.

Keep sperm on ice in a 1.5 milliliter tube and transfer the cytes in filtered a SW into pre rinsed Petri dishes. Transfer 100 to 500 cytes to another 35 millimeter petri dish to perform the injections. Lastly, fertilize the remainder of the cyte clutch as a control for sperm and cyte quality or for other experiments.

To begin the injection procedure, install the needle on the micro manipulator at a 50 degree angle relative to the horizontal plane under a fluorescent dissecting scope with 25 ex oculars, transfer 30 cytes to a polylysine coated dish and filtered a SW to aid in keeping track of injected cytes. Deposit them along a line in an ordered way. Always inject small numbers to minimize the exposure.

Time to polylysine, which tends to deform developing embryos. Use the dark field illumination to render the cytes as translucent as possible. Then with the coarse movement knob, bring the injection needle close to an cyte with fine forceps.Cut.

Open the needle at the level where the tip starts to curve by pulsing with the injector. Verify that red injection mix is flowing out of the needle at 200 x magnification. And with the fine movement knob, gently move the injection needle inside the core of the cyte.

If inserted two superficially, the injected solution will not remain inside the UO site. If inserted too far, the UO site will be destroyed. Inject with one to three pulses.

If the needle is fine enough, inject with continuous flow at constant pressure. Ensure that the injection volume corresponds to one fifth to one third of the volume of a single oocyte following injection. Pull the needle out swiftly to avoid leakage.

Verify that the injected solution remains within the cyte and that after a few seconds, the injected solution spreads throughout the cyte. Move on to the next cyte in line. Keep some unin injected embryos of each series as negative controls to estimate the background fluorescence when scanning for injected embryos fertilize the oocytes as soon as the series has been injected.

Because oocyte quality declines with time, inject and fertilize cytes within one hour. After spawning. Depending on the sperm concentration, add one to five drops of sperm to the UY and swirl the dish.

The fertilization envelope should become apparent on the embryos. After about one minute, allow the embryos to detach from the polylysine coated dish while injecting another series of oocytes. Once detached, transfer the embryos into an aros coated Petri dish.

Remove the embryos from the polylysine coated dish if at all possible. Before the two cell stage. At the two cell to four cell stage, select successfully injected embryos with the aid of a fluorescent dissecting scope.

With a DSR filter, a successful injection is indicated by normal morphology that exhibits a phenol red derived red fluorescent signal. Keep the embryos and filtered a SW in agros coated Petri dishes at 19 degrees Celsius until the desired stage for in vivo imaging, these representative images of bronchos stoma lancia lato embryos were taken after microinjection of mRNAs and coating fluorescent proteins. This first image shows that expression of nuclear EGFP obtained by microinjection of an H two B-E-G-F-P construct can be detected in embryos as early as the 16 cell stage.

This gastro stage embryo is an example for ubiquitous co-expression of nuclear m cherry and membrane targeted EGFP. The nuclear signal results from microinjection of an H two BM cherry construct while fluorescence at cell membranes is obtained by micro injecting an EEG FP CAAX box construct. An optical section of this gastro stage embryo shows how nuclei and cell outlines are highlighted respectively by nuclear targeted mCherry and membrane associated EGFP.

The visual demonstration of this microinjection technique will support researchers in the evolutionary development of biology community to improve existing and to develop novel approaches for individual imaging, gene specific manipulation and stable in afi, an important model for understanding animal evolution.