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Team:Queens-Canada/transformation
From 2010.igem.org
Microinjection
Microinjection takes about a week to learn how to do, and a few months to get highly proficient at. Once experienced, however, the actual act of injection only takes a few minutes. Most universities have at least one microinjection table, and we recommend that teams not comfortable with devoting the resources to training one of their members to microinject ask for assistance from an experienced technician. The actual process involves injecting DNA into the mother’s ovary using a very fine needle under a high-powered microscope.
Below is our standard protocol for performing microinjections as used by Hao Shi. He spent most of the summer practicing and was successful when we started our real injections in August. Learning the microinjection process is best done with the assistance of a live instructor, but these resources may also help you:
The WormMethods link also includes information on techniques for integrating DNA into chromosomes. Since microinjection isn’t perfect, these may still be of use.
Materials
- Linear gene construct or plasmid
- Salmon sperm DNA (filler DNA)
- Inverted microscope with microinjection stage
- Micropipette puller
- Microcapillaries
- M9 microinjection oil
- Injection pads
- Microscope slide with glass cover slip
C. elegans is unusual in that its chromosome duplication is holocentric: the proteins necessary to split the duplicated DNA strands don’t focus on one specific, defined spot, but distribute evenly over the length of the molecule. As a result, we can use just about any DNA format we want to transform worms with, including bacterial plasmids and PCR products. This isn’t perfect; worms have been shown to have up to a 2% chance of loss of artificially-introduced high-copy DNA, compared to a maximum reported rate of 0.5% for chromosomal loss, suggesting that the structure that forms isn’t quite as stable as a natural chromosome. Whether this is structural or if some sequence on the chromosomes assists efficient replication isn’t clear.
Injection Pad Preparation
- Mix 0.2 g of agarose and 10 mL of ddH2O and microwave for 30 s.
- Place 2–4 drops of 2% agarose onto the center of a thin glass slide.
- Flatten the drop with another slide placed perpendicularly on top.
- Allow slides to dry for 5–10 minutes and separate the two glass slides.
- Allow slides to dry over night before use.
Needle Preparation
- Place the microcapillary tube into top slot of micropipette puller and tighten the top knob. # The capillary should be protruding approximately 1.5-2.0 inch from the top ridge.
- Slide up the bottom unit and clamp onto microcapillary with bottom knob.
- Turn on machine.
- Fix machine settings to Temperature = 740 and Pull = 940.
- Close cover and press “start” button.
- The microcapillary tube will be pulled into two needles after a few seconds.
- Remove needle in top clamp and place horizontally onto a piece of clay.
Sample Preparation
- Prepare sample by mixing 30 µL of gene construct, 30 µL of reporter, and 40 µL of filler DNA. Alternatively, if reporter is already included in the construct, then add 30 µL of sample and 70 µL of filler DNA.
- Centrifuge mixture at 13,000 rpm for 10 min to pellet the impurities.
- Pipette 1.5 µL of the supernatant into the un-pulled end of the microcapillary.
- Allow 3-5 min for the sample to move into the needle tip.
Breaking the Needle
- Mount needle onto microinjection apparatus of microscope.
- Prepare a needle-breaking slide by adding a few drops of M9 microinjection oil onto a thin glass slide and covering that with a small glass cover slide. Some oil should squeeze out from the sides of the cover slide.
- Place slide with glass cover slip on stage.
- Locate the edge of cover slip closest to the needle and bring it into focus.
- Use the XYZ axis movement control knobs to bring needle tip into view and in focus.
- Gently touch the edge of the cover slip to the tip of the needle by moving the stage, NOT the needle! This should break the needle.
- Tap the foot pedal for releasing needle contents to check if needle tip is broken properly. # The droplet of water should be approximately 10 times the width of the needle tip.
Microinjection
- Apply a small drop of M9 onto the agar region of the injection pad.
- Flame a pick and dip it in the drop of M9.
- Pick a young adult N2 worm from a plate and transfer it to the M9 on the injection pad.
- Very gently press the worm onto the agar with the pick to prevent it from moving.
- Place the injection pad onto stage. Locate the worm and bring it into focus.
- Bring the needle into focus in the same field as the worm.
- Orient the slide so that the site of injection (the gonads) makes a 45° angle with the length of the needle.
- Use the fine adjustment knob to focus on two rows of cells separated by a clear area beside the row of oocytes. This area represents the gonad of the worm. There are two of such areas (“arms”) in each worm, one in the anterior and one in the posterior.
- Use the Z axis fine control to bring the tip of the needle into focus. The two rows of cells in one arm of the gonads and the needle tip should both now be in focus.
- Slowly bring the worm towards the needle (NOT the other way around) and puncture the side of the worm so that the tip of the needle ends up in the space between the two rows of cells mentioned above.
- Tap the foot pedal to inject DNA into the worm’s gonad. It is important to not bloat the worm.
- Slowly remove the worm from the needle and lift needle away from the slide.
- Repeat steps 5-12 for the other arm of the gonads if possible.
- Flame a pick and transfer the injected worm into a new, labelled plate.
- Store injected worms at 18° C for 3 days and check for F1 offspring displaying the transgenic phenotype. These worms should be transferred onto individual plates for further analysis.
