Protocol for transfection by microinjection into the eggs of the parasite vector snail Biomphalaria glabrata

1. Egg production

Place about 30 adult snails (10 mm diameter) into a 5.5-liter water tank. Place a piece of polystyrene of (3 x 3 cm) in each tank. There is the preferred support of Biomphalaria glabrata for laying its eggs. The snails are fed ad libitum with green lettuce leaves, they can also be fed with dry spirulina to boost reproduction. Maintain water at a temperature of 25 degrees Celsius.

2. Egg collection

Gently pick up several egg layers from the polystyrene with soft holding forceps and place the eggs into a petri dish with natural mineral water (e.g. Volvic) to prevent them from drying out.

Start sorting the eggs under the stereoscopic microscope to choose only the gastrula stage and place them into another petri dish with natural mineral water.

 

3. Preparation of the transfection solution

Material:

a. in vivo JetPEI transfection reagent

b. 10% glucose solution

c. 5% glucose solution

d. Plasmids (dCas9-SunTag-BFP and scFv-DNMT3A-GFP)

e. 0.2 ml microtubes

f. P10 and P200 pipettes

g. P10 and P200 pipette tips

h. Permanent marker

The glucose solution and the in vivo jetPEI transfection reagent are equilibrated at room temperature. 

Prepare 21 µl of each plasmid at a concentration of 78 and 88 ng / µl respectively (for a total volume of 42 µl =equals 3.5 µg of DNA) add the plasmid DNA to a 0.2 ml tube (labeled as Tube A) and mix with 21 µl of 10% glucose solution. 

In another microtube (labeled as Tube B), add 21 μl of 5% glucose solution and 1 μl of in vivo jetPEI. 

 Prepare a third tube (labeled as Tube C) with 21 µl of 5% glucose solution and 0.5 µl of in vivo jetPEI to inject into embryos that will serve as controls. 

Leave the solutions at room temperature while you prepare the microinjection station. 

 

 

4. Preparation of the micro-injection station

Material:

a. Pre-pulled glass micropipettes (1mm diameter)

b. Watch glass

c. Modeling clay

d. 35 mm and 90 mm petri dishes

e. Mineral oil (M5904, SIGMA)

f. Wash bottle with natural mineral water (Volvic)

g. 0.2 ml microtubes

h. 12-well cell culture plate

i. Fine brush

j. Phenol red solution

k. Pasteur pipette or dropper

l. Dissection forceps

m. Soft holding forceps

n. Snail eggs in the gastrula stage

o. Drummond Scientific Nanoject III Programmable Nanoliter Injector

 

Take a pre-pulled glass micropipette and cut it with a scalpel to have a ~ 0.2 mm tip slightly beveled if possible.

Before attaching the micropipette to the programmable nanoliter injector, fill it with mineral oil. If this step is not done, the injector will not work properly. This can be done with a filling needle 

attached to a hamilton syringe of 10 microliters.

When the micropipette is filled with oil, it must be fixed on the injector. For this it is necessary to: 

Slide the chuck and collet onto the glass micropipette, then slide the black O-ring with the seal onto the wire plunger 

With the micropipette attached to the injector, press the [EMPTY] icon until the plunger is fully extended. This step can be done with the footswitch by pressing once [EMPTY] then [STOP] and then proceeding [EMPTY] with the foot switch. A single beep is emitted when the plunger is fully extended.

Fill the micropipette with 3 µl of the control solution or the transfection solution by placing the glass micropipette tip in a 0.2 ml tube with the solution to be injected and pressing the [FILL] icon. It is desirable to fill it at a slow rate, by pressing the [FILL] icon for a few seconds, then the [STOP] icon to allow the sample to equilibrate before pressing again the '[FILL] icon.

Note: The piston continues to extend or retract until the [STOP] icon is pressed, or until the fully extended or fully retracted position is reached.

 

5. Microinjection

Place a watch glass into a 35mm petri dish and secure it on one side with modeling clay to form a slope. Use soft handling forceps to transfer an egg mass and lay it on the slope side of the watch glass so that the egg mass is in a sloping position.

Remove excess water from the eggs with absorbent paper. Rehydrate if necessary with a fine brush to improve the visibility of the embryos. To inject the sample, return to the operating mode screen by pressing the [EXIT] icon, then select the injection mode by pressing the [INJECT] icon. Set the injection volume to 30nL and the flow rate to 20nL per second using the icons [+] and [-] respectively. Press the [INJECT] icon to inject the sample. 

Inject 30nL of the microinjection solution into each egg. Place the microinjected egg masses in a 12-well cell culture plate and note with a marker whether they were microinjected with the control solution or with the solution containing the plasmids.

We colored the injection solution with red phenol to facilitate the visibility in this video.

Monitor the expression of the plasmids

Monitor the plasmids expression 72 h after microinjection in a contrast / fluorescent microscope or in a fluorescent stereo microscope. Then sort the fluorescent snails and perform a second micro-injection with a solution containing 10 µl of single guide RNA (at a concentration of 2ng / µl), add 0.5 µl of in vivo jetPEI reagent and 10 µl of 5% glucose solution. 3 days after the second microinjection, collect the hatched snails in a 1.5 ml tube containing 25 µl of lysis buffer for DNA and RNA purification.

In this photo produced under a confocal microscope we washed a veliger larva in PBS solution, then we fixed it with 4% paraformaldehyde solution and then we placed it in a slide with two drops of the Dako fluorescence mounting medium. 

96 after the transfection we can observe the expression of the green fluorescent protein, the blue fluorescent protein and the co-localization of both proteins. 

This protocol is used to perform DNA methylation changes in a target gene. This transfection protocol can be used with other plasmids, with small interfering RNAs, or with messenger RNAs.

Produced at IHPE (http://ihpe.univ-perp.fr)