The overall goal of this procedure is to generate transgenic worms via biotic or gene gun transfection. First large quantities of worms are grown, then gold particles are coated with DNA, and worms are bombarded, transgenic, progeny, then isolated and rescued, and new transgenic lines are established. Hi, I'm Daniel Owa from the laboratory of Dr.Alfred Fisher in the Medicine Department at the University of Pittsburgh.
Today we'll show you a procedure for generating transgenic worms. We use this procedure to study gene regulation by transcription factors. So let's get started.
Egg plate preparation begins by pouring a mixture of 2%agar in NGA medium supplemented with Nystatin and strep mycin. In 10 to 50 centimeter plates allow the plates to dry at room temperature. Next, autoclave, 400 milliliters of LB media in a 500 milliliter bottle and 40 milliliters of LB media in a 250 milliliter flask.
Also autoclave an empty 500 milliliter bottle for 20 minutes. Once autoclave, add streptomycin to the 40 milliliters of LB medium to a final concentration of 200 micrograms per milliliter. Then inoculate with e coli strain.
OP 50 dash one. Allow the culture to grow overnight at 37 degrees Celsius. The next day, place the yolks from 10 chicken eggs into the sterile 500 milliliter bottle.
Bring the volume up to 400 milliliters with LB medium and shake to form a uniform solution. Incubate the solution at 60 degrees Celsius for one hour. Once the yo lb mixture has called to room temperature, add the 40 milliliters of ELI culture to the bottle and shake.
Well then pipette five to eight milliliters of the culture onto the prepared NGA plates. Allow them to dry overnight at room temperature the following day. Pour off any remaining liquid and dry for an additional night at room temperature.
At this point, the plates can be stored at four degrees Celsius for one to two months or immediately seeded with worms. To expand a DP 38 CL agains population had a concentrated e coli OP 50 to six centimeter NGA plates containing recently starved DP 38 worms and incubate. The worms use alkaline hypochlorite solution to isolate the eggs from the DP 38 worms.
The procedure is described in the accompanying protocol. Once the eggs have been isolated, resus, suspend them in s basil or M nine buffers. Add more than 10, 000 eggs to each prepared 10 centimeter egg plate and allow the worms to grow at 20 degrees Celsius for seven to 10 days.
Once the worms have begun to clear the food, the plates are ready to be bombarded. To coat gold particles with DNA For bombardment, add 60 milligrams of gold particles to a 1.7 milliliter tube. Then add 70%ethanol and allow the gold to soak for 15 minutes.
Spin the tube briefly to pellet the gold particles. Decant the ethanol. Then wash the gold particles three times in sterile water.
Resuspend the gold in one milliliter of 50%sterile glycerol. The gold particle stock solution can be stored for months at four degrees Celsius. Next, collect the worms by floating them from the egg plates using s basil or M line buffers.
Then transfer them to a 50 milliliter conical tube. Allow the worms to settle to the bottom of the tube. Then aspirate all but two to three milliliters of the buffer from the tube using the remaining buffer.
Transfer the worms to a 10 centimeter unspotted NGA plate that has been called on ice evenly. Distribute the worms on the entire plate. Then allow the plate to dry completely on ice.
This will prevent the worms from clumping during the drying process. It is important that the worms are evenly distributed and that the plate is completely dry before the bombardment procedure. To prepare the DNA coated gold particles, add 50 microliters of resuspended gold particles to a 1.7 milliliter tube.
Spin down the gold particles and remove the supinate. Then add a mixture of 10 to 15 micrograms of purified, circular or linearized DNA of interest and 20 microliters of freshly prepared 0.1 molar sperm aine. Finally, while vortex in the tube gently had 50 microliters of 2.5 molar calcium chloride, incubate the mixture on ice for 30 minutes with periodic mixing to keep the gold particles in suspension.
After 30 minutes, spin the gold particle mixture. Briefly aspirate the supernatant wash with 300 microliters of 70%ethanol, followed by one milliliter of 100%Ethanol finally resuspend the DNA coated gold particles in 170 microliters of 100%ethanol. For each bombardment rinse seven macro carriers which will hold the gold particles in two propanol and then allow them to dry at room temperature.
Vortex the DNA gold mixture. Then pipette 20 microliters onto the center of each macro carrier. Let the ethanol evaporate.
Insert the rupture disc into the Hector adapter and secure the adapter into the bombardment apparatus. Then place the seven macro carriers into the holder and use the supplied tool to seat them. Next place a Hector stop screen and the bottom onto the holder.
Flip over the holder so that the gold spotted side of the macro carriers is facing down. And place it on the second shelf from the top. Align the holes in the top of the macro carrier holder with the outlets of the Hector adapter.
Remove the lid from the NGA plate coated with worms and place it on the lowest shelf in the bombardment chamber. Completely open the vacuum flow rate knob and press and hold the vac button until the chamber reaches 26 inches of mercury. Then switch to the hold position To maintain the vacuum, press and hold the fire button until the disc ruptures.
This will occur when the pressure reaches 1, 350 PSI and can take five to 20 seconds. Release the button, release the vacuum from the chamber by switching to the vent position and remove the plate. Turn off the vacuum in helium.
Then press fire several times until the helium line is clear and the helium gauge reads. Zero PSI finally turn off the PDS 1000 system. Now that the worms have been transformed, they can be recovered and incubated after bombardment.
Place the plate at 20 degrees Celsius for 20 minutes. Then float the worms from the plate with 10 milliliters of S basil or M nine buffer inoculate, 2010 centimeter spotted NGA plates with 0.5 milliliters of the worms in buffer. Then leave the plates at room temperature for approximately two weeks.
Screen for rescue of the UNC one 19 phenotype by the transgene and generate one individual line from each 10 centimeter plate that contained the rescued worms. The success of the protocol with regards to obtaining transgenic animals depends on the particular transgene. For promoter GFP Reporter transgenes, we have obtained up to 20 lines.
A more typical result is three to 10 lines. Up to 30%of the transgenic lines are integrated lines, but this is random and we'll perform multiple bombardments on a single day if an integrated line is particularly desired. Shown is a representative 10 centimeter plate shown onk one 19 mutant worms rescued by transgene bombardment.
The rescued worms can be identified by the larger size and much improved mobility relative to the non transformed. On one 19 mutants. Isolation of rescued worms transformed with a far one GFP transgene results in GFP expression.
This transgene expresses GFP strongly in the intestine and hypodermis of transgenic worms. This particular transgene is integrated into the worm chromosome and 100%of the progeny express the transgene. We've just shown you how to perform bio holistic bombardment of sea elegance when doing this procedure.
It's important to grow plenty of worms and use fresh permitting. So that's it. Thank you for watching and good luck with your experiments.
