The overall goal of this mutagenesis protocol is to use a simple LED set-up to mutagenize the genomic DNA of C.elegans that express blue light-dependent, reactive oxygen generator proteins. The two main advantages of this technique are one, the mutagenesis protocol is very simple and do not require the toxic chemicals. And two, the mutagenesis generator proves after a long mutation.
I first have the idea for this method, and I realize that ROS-generating proteins have never been used to induce heritable mutations. For this experiment, connect the controller of the LED illuminator device to a digital function generator using a BNC cable. Using a custom holder, secure the LED lights 10 centimeters above the stage.
Now, partially cover the set-up to limit exposure of the blue light to the surroundings, but not so much that heat will accumulate on the stage. A custom made, hard plastic cover with an open top and bottom is employed here. During the LED's operation, always wear laser-protective glasses or sunglasses, because the light is very bright.
Now, switch the lever of the LED controller to internal, and set it to 65%of its maximum power. Using a photometer, adjust the light's power if necessary, so that on the stage, it is 2.0 milliwatts per square millimeter during continuous illumination. For this protocol, maintain the light-sensitive strain in the dark under standard conditions.
Unnecessary light exposure should be avoided, but routine halogen lamp use for passage, does not result in mutations. Because accidental mutation is a concern, freeze aliquots of the strain when they are first obtained. For the mutagenesis, prepare a filter paper to fit a 60 millimeter plate with an approximately 25 millimeter square hole at the center, and place it onto an unseeded plate.
Then, distribute 100 microliters of 100 millimolar copper chloride evenly over the filter paper to keep the worms from leaving the square hole. Now, from a plate of worms, that are about 12 hours after their L4 stage, pick the gravid, young adult hermaphrodites and put them into the center square on the plate. Next, turn on the LED and the function generator.
Then, switch the LED controller to external to control it with the function generator, and set the power appropriately. From the function generator, produce a sine wave at 4 hertz. Now, transfer the plate to the stage and illuminate the worms with blue light for 30 minutes.
Immediately after the treatment, transfer all the healthy looking worms to a seeded plate. These worms will appear uncoordinated after the light treatment, but they will recover. Keep the light-treated worms in the dark.
Later, check for abnormal morphologies or behaviors in the adult F2 progeny. A forward genetic screen was performed using the described technique for 60 F1 plates, corresponding to 120 mutagenized haploid genomes. Eight of the 60 F1 plates with F2 worms displayed visible phenotypes, such as dumpy body shape, uncoordinated movement, or adult lethality.
The progeny of these F2 worms retained these observable phenotypes. The number of visible mutants from this screen suggests that the strain and set-up are working properly for optogenetic mutagenesis. The mutagenesis procedure can be done within an hour.
Please enjoy the fast and safe mutagenesis method.
