Background of C. elegans Microinjection

Caenorhabditis elegans (C. elegans) is an important model organism used for studying animal genetics, ontogeny, and ethology. It can help us to study the mechanism of action from molecular and cellular level to system biological level in regard to the corresponding life cycle. Microinjection technology provides a capillary glass tube with a tip diameter of millimeters for the injection of nucleic acids into the worm’s gonad that is subsequently absorbed by the egg cells and generated into the form of extrachromosomal arrays. C. elegans microinjection is the core technique in the C. elegans research paradigm. It is widely used in studying gene expression, gene function and genetic interactions inside the body of c elegans.

Workflow of C. elegans Microinjection as shown in the figure

C. elegans Microinjetion Protocol

Step 1: Preparation of agar plates

Drop 50ul of 2% hot agarose solution on a 24 × 60 mm glass coverslip and gently put another coverslip on top (beware of the air bubbles). After the flattened agarose solidifies (~5 min), you can remove the coverslip on top by gently sliding. Let the agar plate dry overnight at room temperature or bake it at 80℃ for 1 hour. Stack the plates for later use. 

Figure: Preparation of the agar plate[1]. A. Mount the coverslip on a clean working stage with 2 tapes. B. Drop 50ul of 2% hot agarose solution in the middle of the coverslip C. Place another coverslip on the agarose drop and gently press it to make it flattened. Remove the coverslip after the agarose solidifies. 

Step 2: Preparation of needles

The needle is the key to successful C. elegans microinjection. RWD micropipette puller helps with generating two needles with stable and consistent performance. It is recommended to use RWD capillary glass tube for pipette pulling to fill up the tip of the needles quickly with stock solution. As such, the air bubbles can be discharged. In general, the tip diameters are down to 1μm and the cone length is around 5~7mm.

Step 3: DNA preparation and fill a needle-loading pipette

According to the experimental requirements, choose the substances for stock solution. For example, DNA, RNA, proteins and other substances. The purity of stock solution is one of the important factors affecting injection efficiency. Use a small and thin chemical dropper to introduce the stock solution into the pipette through the tip of the needles. After several minutes, observe the tip to see if there are any air bubbles. 

Step 4: Breaking

Place the coverslip on the agar plate filled with stock solution. Move the RWD Micromanipulator to smash the needle against the edge of the glass slide. When the tip is broken, the vacuole will leak out. This method allows easy penetration of the needle into the worm. 

Step 5: Mounting worms

Young gravid worms are commonly selected for microinjection as their gonads are fully developed at this stage in which the worms are readily subject to DNA transfection, resulting in the generation of transgenic offspring. Use a pick to place the worm on the agar plate. Adjust the position so that the gonad is exposed outside. Add a few drops of halocarbon oil to fully cover the worm.

Figure: Worm mounted on the agar plate and prepared for injection. The arrows show the gonads selected for injection. The proportion is 50μm[1]. 

Step 6: Microinjection

Place the agar plate on the mechanical stage. Find the worm under the microscope and focus on the gonad by adjusting the condenser lens. Use the RWD Micromanipulator to insert the needle into the gonad. Switch on the RWD Nanoliter Microinjection Pump to start injecting. 

Figure: The flow of injection mix before (1) and after (2) microinjection. The arrow marks the gonad areas where the injection mix arrives^[2]. 

Step 7: Worm recovery

Add a few drops of buffer solution to the injected worm under the stereomicroscope. After 2-5 minutes, the worm will become active again. It should start swimming and moving its head from side to side. By then, you can transfer it back to the cell culture plates for regular cultivation at 20℃.

Precautions^[2]

Agar plates: If the worm dies quickly on the agar plate, it is an indication of the plate being too dry. In this case, it can be replaced by another plate with a thinner agar layer since the agarose is mainly used for absorbing water from the worm; If the worm cannot stick firmly on the plate, it is an implication of the plate being too wet or too thin. In this case, it is advised to put it in the oven for a period of time or stick the worm on the plate before adding the halocarbon oil. 

Substances for injection: Keep the DNA solution well mixed and centrifuged before the injection to prevent the needle tip from getting clogged. The concentration of DNA should be kept under 200mg/L since highly concentrated DNA solution will produce toxins and lead to gene overexpression. 

Injection orientation: The needle and the gonad should be positioned at a sharp angle. It is recommended to place the needle horizontally or at a 15°angle to the head or tail.

Sample used in the experiment: Make sure the injected worms are well-fed and healthy.

High death rate of injected worms: For worm recovery, if the time taken is too long or too short, it may be due to the needle being too big or the injected DNA being contaminated. To address this issue, the thinner needle could be the remedy. Also, try injecting only one gonad at a time. If the microinjection procedure has been done properly but no transgenic worms arise, the possible reasons may be lethality induced by genetic transformation or the injected nucleic acids being polluted that requires purification again. 
 
References:
[1] Matthias Rieckher and Nektarios Tavernarakis. Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection [J].Bio Protocol, 2017, 7(19): e2565.
[2] Krishna S. Ghanta et al., Microinjection for precision genome editing in Caenorhabditis elegans[J].Star Protocols, 2021, 2(3): 100748.