The overall goal of this procedure is to perform two different mutagenesis protocols that are commonly used to generate mutants and nematodes, such as ceno, hepatitis elegance, and prisca. Pacificus mutagenesis is a powerful tool to uncover the function of genes. This is accomplished by first preparing cultures with large numbers of worms at the last larval stage before adulthood, with which the mutagenesis will be performed.
The next step is to mutagen the worms using two alternative procedures, ethyl methane, sulfonate treatment, or sore and mutagenesis in conjunction with ultraviolet radiation. The third step of the procedure is to transfer healthy looking worms to agri plates, freshly seeded with bacteria, which is the food source. The final step of the procedure is to screen for mutant phenotypes of interest in the first, second, or third generation.
Hi, am Jore from the lab of Dr.Andre Perez, the Silva in the Department of Biology at the University of Texas. At Arlington, the T-M-P-U-V and the EMS Mutagenesis procedures help answer key questions in the field of genetic research, such as which genes are involved in biological processes that has an important role to play during development. Hi, my name is Manish Phar, though the mutagenesis methods demonstrated will be for Pristi Onca S Pacificus, they can be very commonly employed for other experimental models like the nematode sea elegance, and even vertebrates like zebrafish.
So let's get started. Our pick for transferring worms from one location to another is made from 30 gauge, 90%platinum, and 10%iridium wire. Although some researchers may prefer slightly different metal compositions to make a pick a pasture pipette is also used to hold the pick.
Start by breaking the extended tip of the pasture pipette to a required length. Cut about three to four centimeters of wire and place 0.5 centimeters of it. Inside the tip of the pasture pipette seal the wire into the glass over a bunsen burner.
The length of the wire protruding from the glass is about three to three and a half centimeters, but can vary according to individual preferences. Flatten the end of the protruding wire using pliers. Then bend the flatten portion upward to form a scoop.
Finally, smooth the sharp edges of the pick with sandpaper to prevent damaging the worm or the agar. To pick worms, sterilize the wire of the pick on a flame, and then drag the flattened tip along a bacterial lawn on an NGMP tree plate to coat the tip with thick sticky bacteria. Be careful not to puncture or damage the agro surface using a stereo microscope very lightly.
Brush the sticky tip against the worm to be picked until the worm sticks onto the bacteria on the pick. Once the worm is on the tip of the pick, transfer immediately to the new plate by touching or sliding the tip of the pick against the bacterial lawn of the new plate. Care must be taken not to damage the agro surface of the plate where the worms may crawl into the holes and become difficult to retrieve.
The worm should crawl off the pick when you touch or slide the tip of the pick against the bacterial lawn. The worm should not stay on the pick for too long or it might desiccate. We will demonstrate EMS mutagenesis on four to five six centimeter diameter Petri plates of prisca pacificus worms at the third larval juvenile stage.
Wash the worms off each plate using two to three milliliters of sterile M nine per plate. There should be a few hundred worms. Collect the worms in a sterile disposable 15 milliliter centrifuge tube.
Centrifuge at 1500 Gs for five to seven minutes or until the worms form a pellet. Aspirate the liquid and use two milliliters of M nine to resuspend the worms wearing double gloves and working in a fume hood at 20 microliters of EMS to a tube containing two milliliters of M nine and shake to dissolve it. Then add this to two milliliters of worm suspension and swirl gently.
The final concentration of EMS will be 47 millimolar. Place the centrifuge tube with paraform on a low speed rocker in the fume hood and let the worms incubate for three and a half hours. At the end of the three and a half hour incubation, place the tube in a vertical position and incubate until the worm sink next, aspirate the supra natant and discard it into a beaker containing one normal sodium hydroxide To inactivate the EMS in the supernatant.
Each time before removing your hands from the fume hood, be sure to rinse them also in one normal sodium hydroxide. To inactivate the EMS, add five milliliters of M nine to the worms. Invert the tube 25 to 30 times to wash the worms and centrifuge at 1500 Gs for five to seven minutes or until the worms form a pellet after centrifugation, aspirate the supernatant and discard it into the one normal sodium hydroxide peaker.
Repeat this washing step of adding M nine centrifusion and removing the snat at least three times. After the final wash, resus suspend the worm pellet in a minimal amount of M nine, pipette the worm suspension onto NGM plates containing a lawn of OP 50 bacteria. After letting the liquid dry for a few minutes, pick and transfer healthy looking J four larvae to fresh seeded plates.
Let the mutagen worms self fertilize about eight to 10 days later, use a stereo microscope to screen the F two generation phenotypes for excessive mutants of interest. This procedure for mutagenesis will be demonstrated on ppac pacificus worms from four to five six centimeter diameter Petri plates collected into a 15 milliliter centrifuge tube. Add 10 milliliters of M nine to the worm pellet.
Invert 20 times to wash the worms and centrifuge at 1500 Gs for five minutes. Discard the supernatant after centrifugation. Repeat the washing of the worms with M nine for a total of three times after the final wash, aspirate the liquid and resuspend the worms in about 10 times their volume of M nine.
Then add 4 5 8 trimethyl SOREIN or TMP from a three milligrams per milliliter stock to get a final concentration of 30 micrograms per milliliter. TMP incubate the tube in the dark at room temperature on a low speed rocker for 15 minutes. After the incubation, set the tube vertically for 10 minutes to let all the worms sink for this video, the tube is shown uncovered, but the TMP solution should not be exposed to light.
Because this chemical is light sensitive, then use a past pipette to remove the worms from the bottom of the tube and transfer them onto an unseated NGM plate. Keep the plate in the dark until the excess TMP is absorbed into the plate. Once the TMP is absorbed into the plate, the worms can be treated with ultraviolet light.
One of the most critical steps in the TMP UV neurogenesis procedure is to have the right distance between the long wave UV source and the plate. So we have to adjust the distance a couple of times to get the right intensity of UV light falling on the plate. The intensity should be recorded by the intensity meter, and it should be 500 micro watt per centimeter square.
Proper eyewear should be used during this step because of the potential risk of the UV light. Using a UV intensity meter. Determine the distance from the long wave UV source that would result in an intensity of 500 micro watts per centimeter squared.
Next, remove the aluminum foil and lid and place the plate containing the TMP treated worms at a distance from the lamp so that the intensity on the plate is 500 micro watts per centimeter squared. Expose the plate to the longwave UV radiation for 50 seconds cover and leave the worms in the dark for five hours at room temperature. After five hours, pick and transfer healthy looking J four larvae to fresh seeded plates.
Finally, screen the F1, F two or F three generation for phenotypes of interest using a stereo microscope. Shown here are some of the ppac pacificus mutant phenotypes identified in the F two generation after either EMS or T-M-P-U-V mutagenesis. The top two panels in these images are examples of a wild type hermaphrodite and a wild type male.
Each mutant has a characteristic phenotype that is easily distinguishable from the wild type animal. Panel C is an example of a dumpy mutant. Panel D shows an uncoordinated mutant and panel E is of a transformer mutant Once mastered, the EMS mutagenesis can be done in four and half hours, and the TMP uuv mutagenesis can be done in five and a half hours if performed properly.
This time period also includes the incubation time. Don't forget that EMS and TMP are extremely hazardous and while working with them precautions such as wearing a lap coat using double gloves, the use of a fume hood and safety goggles must always be taken. Thanks for watching and good luck with your experiments.
