Sea elegance is an incredibly useful model organism due to both our knowledge of its cell lineages as well as the wealth of information produced from having sequenced and analyzed its entire genome. In this video, we demonstrate the process of micro injecting plasmids into sea elegance and the selection of transgenic offspring using fluorescent microscopy as a transparent and multicellular animal, sea elegance is ideal for visualizing transgenes expressing fluorescently labeled proteins in vivo. Hi, my name is Laura Berkowitz and I'm a research associate in the Caldwell Lab in the Department of Biological Sciences here at the University of Alabama.
And I'm Adam Knight, also from the Calwell Lab at the University of Alabama. Today I will be showing you how to prepare for and perform Plasmin Microinjection to generate transgenic sea elegance, And I'll be showing you how to select and maintain transgenic seal elegance lines. So let's get started.
Before we begin the microinjection, we have a few preparation steps, which I'll show you now. First, prepare the plasmid DNA to be injected, two plasmids are required, one that encodes the tissue specific expression of our gene of interest, and the second that provides a selectable transformation marker. Mix the plasmids together in a one-to-one ratio of 50 nanograms per microliter.
Each next, prepare auger pads, which will be used to immobilize the worms for injection. To make the auger pads first set out several 22 by 50 millimeter cover glasses on the benchtop. Using a pasture pipette place a drop of melted 2%agros on one cover glass, and immediately place a second cover glass over the drop at a 90 degree angle to the first.
Let the aero solidify for a few seconds and then remove the top cover glass. The diameter of the flattened disc of aero should be between 15 and 20 millimeters. Like a pet, allow the pad to air dry completely for several hours or overnight.
Once they are dry, store the pads in a cover glass box at room temperature. Next, prepare the needle loaders for the DNA solution. To do this, take a hundred microliter capillary tube and heat it in the middle of an open flame and rapidly pull it to approximately twice its length.
Once the capillary tube cools, snap the two halves apart. Stockpile 10 to 20 needle loaders for injection. You can store them upright in a micro centrifuge tube.
Brack, be careful. The needle loader points are very sharp. Now make the micro injection needles.
These needles are made from a special capillary tube that contains an internal glass filament. This filament serves as a wick for the DNA solution. To make the needles we use a Niki PP eight 30 Polar With a heat setting of 24.8, several needles are pulled at a time for storage.
Multiple needles can be mounted on a strip of modeling clay. Small shards of cover glass will be used to break open the pulled needle tip. These shards are prepared by wrapping an 18 by 18 millimeter cover glass in a Kim wipe and gently applying pressure until it breaks into several pieces.
Use shards that are approximately three by four millimeters in size. In addition to preparing the DNA and needles, prepare about 50 to 100 properly staged. Two wild type worms for each expression vector construct to be I injected.
We are now ready to set up the microinjection microscope. To begin setting up the microscope, open the main valve on the helium tank that is connected to the microinjection needle arm and holder. Close the regulator to allow the gas to enter the line.
Bringing the pressure to 35 PSI the gas can be released using a foot pedal. Next, insert a needle loader into a standard mouth pipetter tubing and drop a small amount of the plasmid mixture approximately one microliter. Now gently insert the loader all the way through the microinjection needle until resistance is felt.
Expel the DNA solution by gently blowing and then remove the loader. Once the DNA is loaded, insert the needle into the microinjection arm, being careful to place it within the small internal rubber gasket and then clamp it to the arm of the manipulator. Next place a needle breaking shard of cover glass on one edge of an auger pad.
Cover the shard as well as the whole surface of the auger pad with hallow carbon oil. Then place the auger pad on the stage of an inverted microscope. After the auger pad is in place, position the needle in the center of the viewing field using the Forex objective and bright field illumination, but do not yet lower it into the oil.
Next position the inner edge of the shard of cover glass in the center, and then focus on it. Lower the needle into the oil, bringing it into the same focal plane as the shard of cover glass. Raise the magnification by switching to the 40 x objective, then refocus on the edge of the shard and reposition the tip of the needle if necessary.
Now break Open the tip of the injection needle. Gently nudge the needle against the edge of the cover glass shard, while at the same time applying a short pulse of gas via the foot pedal. The needle will be sufficiently broken open.
When small droplets of liquid escape the end of the needle excess liquid flow due to too large, an opening is not desirable as it will kill the animals. Once the needle is ready, we begin the microinjection. Next, remove the auger pad from the stage by raising the needle up.
But do not change the X or Y axis position. Slide the stage out from underneath the needle. Remove the auger pad and place it onto a dissecting microscope.
Transfer a word onto the pad below the surface of the oil. If the worm wriggles gently stroke it until it contacts the auger pad, the moist worm should adhere to the dry agro. Quickly place the auger pad back onto the stage, centering the worm in the field of view.
Then lower the needle into the same plane of focus as the worm. Switch the filters on the inverted scope to Hoffman illumination. This contrast filter allows morphological detail of the worm to become visible.
Using the 40 x objective, focus on the grainy sensorial center of the worm gonad below the honeycomb pattern of the germ nuclei. Choose whichever gonad is positioned on the side of the worm facing the needle.Fine. Tune the position of the needle until the tip is also in focus.
Gently insert the needle into the center of the gonad and apply a brief pulse of gas pressure to expel a droplet or two of DNA into the gonad arm. You should observe a wave of liquid spread across the distal goad with a lot of practice and skill. You can hit the goad center every time.
Keep in mind that it's important to work quickly while injecting a worm. As it can easily desiccate. The entire process should ideally take less than one to two minutes.
Once the injection is completed, remove the auger pad from the stage and place it on a dissecting scope. Apply one microliter of M nine buffer directly onto the injected worm. This might entail submerging the pipette tip below the surface of the halo carb oil.
The buffer should rehydrate the worm and it will then float off the aeros pad surface, transfer the worm to a regular plate using a worm pick. Now I will hand off the injected worms to Adam and he'll show us the selection and maintenance of transgenic lines. The injected worms are transferred to individual fresh seeded 60 millimeter plates and allowed to reproduce.
These worms represent the P zero generation. Typically injected animals are incubated at 20 degrees Celsius for two to three days until the offspring appear. Once the offspring appear, we are ready to begin screening for transgenic worms.
The F1 offspring are observed for evidence of the transgenic marker. In this case, the selectable marker is fluorescent, so we can screen using a fluorescent stereo microscope. Each fluorescent carrier F1 animal is separately cloned onto a new plate.
The injected P zero may only produce one to five fluorescent offspring among all of its F1.Allow the F1 hermaphrodites to reproduce at 20 degrees for two to three days until the offspring appear. Once the F two generation is ready, they're observed for transgenic animals as well. F two animals that have inherited and expressed a transgenic marker are considered a stable line.
Each independent stable line should be maintained as a separate line of worms propagate each line by transferring several transgenic animals to a new plate at each generation. This must be performed using fluorescent stereo microscope. If the selectable marker is fluorescent to control for the variability in the gene copy number, generate a minimum of three independent stable lines per construct injected.
We've just shown you how to micro inject expression, vector constructs, and see elgan and to select for stable lines. So that's it. Thanks for watching and good luck with your experiments.
