Obtaining viable embryos can be the most trying part of generating transgenic fish. Putting in time up front to maximize survival, and to be able to get embryos on demand will be well worth your time!
General considerations
If your cichlid species of choice is not a year-round spawner, get to know the conditions that will elicit egg laying.
Even year-round spawners have ovarian cycles that are weeks-long (A. burtoni’s is ~4 weeks). With such long cycles, any given female is unlikely to be gravid at the time you are ready to inject the embryos. Therefore, I have a large cohort of females isolated from males, so that I increase the odds of getting a spawning female. Additionally, being able to identify those females that are ready to spawn allows me to collect eggs when I see signs that a female will lay eggs. For A. burtoni, this includes a distended abdomen packed with eggs, a protruding genital papilla, and aggressive behavior toward other females.
Before doing any injecting, ensure that you can collect fertilized eggs just after spawning and raise them to juvenile stage. This can allow you to see whether fertilization is efficient, and whether there are any other factors necessary to get the eggs to survive. Luckily for us, A. burtoni appears to survive just fine outside the female’s mouth.
Setting up
Fish housing
House a single stud male and a cohort of females separately by sex. I keep one male and 7-15 females in a 168 x 46 x 30 cm tank, separated by a barrier. (I have also transferred females into a stud tank and gotten successful spawnings, but this involves more work and may disturb the fish.)
Injection setup
We connect a compressed air tank to a MPPI-3 pressure injector with back pressure unit, which delivers a specific duration of air pulse. This connects to a pipet holder mounted on a micromanipulator. The eggs are held in place in a custom-molded agarose chamber. We have 3D printed a mold which we place into 2% agarose dissolved in fish tank water. See below for link to the 3D printing file. When we remove the mold, wells that are the size of cichlid eggs are left behind & covered with fish tank water.
Egg mold. An entry-level 3D printing system using ABS plastic may be used to print the mold with the file at the following links.
.stl file (commonly used format, but an older version of the mold)
.ipt file (newest version of our mold but a filetype that can't be used on all printers)
Modify these files as needed to create wells that are appropriately sized for your species' eggs. The well sizes vary within our species and so we systematically vary well sizes across the mold to ensure snug fits of eggs into the wells. You may want to have a wide variation in well sizes in a first version of your mold to allow determination of the optimal sizes.
Injection needle production
Materials:
First, set the pipette puller to an optimal heater voltage and solenoid current for your needs. We found that setting it to 14.9 units of heater voltage and the solenoid current to 4.7 amps DC regulated worked well for us. Insert the capillary glass tube into the machine and wait for the needle to drop. Wait a minute to allow the glass to cool; it can get really hot! This is where it gets to be less than perfect. The pipette puller is made to produce needle tips within 1-5 microns in diameter. In order for the needle to break through the chorion, it should be between 10-15 microns in diameter.
Hold the kimwipe gently in one hand and the needle in the other. With very light pressure, tap the needle onto the wipe. You should feel a very small snap. To make sure you’ve broken it to a satisfactory diameter, measure the diameter of the needle with a bright-field microscope fitted with a reticule.
**KEY point: We discard needles >15 microns as they result in high levels of death. Screen these for size under a microscope and ensure the outer tip diameter is low.
Note: The first few tries might result in a needle that is broken to 20 or more microns in diameter. This is expected! It takes some practice to get the right amount of pressure when tapping the needle on the Kimwipe.
Day of injections
Remove the barrier separating the fish and watch for spawning. I typically give the fish ~15 minutes, and if there is no sign of spawning, I try another day. If a female does spawn, note the time at which she first has eggs in her mouth. Embryos will be collected 30 minutes from this timepoint.
Prepare for injections. Mix the RNA/DNA/marker to be injected (as described in the Tol2/CRISPR sections), and backfill needles with a gel-loading pipet tip. I put ~1 µL into each of ~3-4 needles so that they can be rapidly switched out if one breaks.
Collect ~800 ml of water from the tank in which the spawning is occurring. Add methylene blue antifungal (final concentration, 1 mg/liter; Sigma) to the water, and then pipet 6 ml into each well of several 6-well plates. Plan to inject as many eggs as possible—usually 30-100 is possible—based on number laid, time until cell division, and speed of injection. Also use ≥10 embryos as uninjected controls. The survival rate among these embryos will indicate the viability of the brood and the quality of raising conditions. Any increased death above this rate can be attributed to the injections.
Injecting embryos
Fertilized eggs are collected ∼30 min after spawning, and injected during the single-cell stage.
Transgenesis mixture:
We usually inject 2-3 pulses @ 2.8 ms, 40 psi (~1 nL per pulse).
Each injected egg was allowed to develop alone in a well of a 6-well culture dish (Greiner Bio-One).
This approach also allows us to track the progression of individual embryos. Try systematically varying injections and seeing whether survival/transgene uptake/etc is improved.
Care for injected fish
At ∼12 d postfertilization (dpf), just before the yolk is fully consumed, injected larvae were transferred to 1.5-L tanks, and then to 32-L tanks at ∼4-6 weeks of age.
At ~4-6 weeks post-fertilization, I take a fin clip, and PCR genotype (suggested protocol in appendix) for the presence of the transgene. In my experience, only those fish carrying transgene in their tail end up passing it through the germline.
Mate transgene carriers to wildtypes, and then check for transmission of transgene in G1 fish, using PCR or other method of verification. If it transmits, then congratulations, you’ve got a stable line! Now test whether it faithfully recapitulates expression in your targeted cell population, and see whether you get the phenotypes you’re looking for!
General considerations
If your cichlid species of choice is not a year-round spawner, get to know the conditions that will elicit egg laying.
Even year-round spawners have ovarian cycles that are weeks-long (A. burtoni’s is ~4 weeks). With such long cycles, any given female is unlikely to be gravid at the time you are ready to inject the embryos. Therefore, I have a large cohort of females isolated from males, so that I increase the odds of getting a spawning female. Additionally, being able to identify those females that are ready to spawn allows me to collect eggs when I see signs that a female will lay eggs. For A. burtoni, this includes a distended abdomen packed with eggs, a protruding genital papilla, and aggressive behavior toward other females.
Before doing any injecting, ensure that you can collect fertilized eggs just after spawning and raise them to juvenile stage. This can allow you to see whether fertilization is efficient, and whether there are any other factors necessary to get the eggs to survive. Luckily for us, A. burtoni appears to survive just fine outside the female’s mouth.
Setting up
Fish housing
House a single stud male and a cohort of females separately by sex. I keep one male and 7-15 females in a 168 x 46 x 30 cm tank, separated by a barrier. (I have also transferred females into a stud tank and gotten successful spawnings, but this involves more work and may disturb the fish.)
Injection setup
We connect a compressed air tank to a MPPI-3 pressure injector with back pressure unit, which delivers a specific duration of air pulse. This connects to a pipet holder mounted on a micromanipulator. The eggs are held in place in a custom-molded agarose chamber. We have 3D printed a mold which we place into 2% agarose dissolved in fish tank water. See below for link to the 3D printing file. When we remove the mold, wells that are the size of cichlid eggs are left behind & covered with fish tank water.
Egg mold. An entry-level 3D printing system using ABS plastic may be used to print the mold with the file at the following links.
.stl file (commonly used format, but an older version of the mold)
.ipt file (newest version of our mold but a filetype that can't be used on all printers)
Modify these files as needed to create wells that are appropriately sized for your species' eggs. The well sizes vary within our species and so we systematically vary well sizes across the mold to ensure snug fits of eggs into the wells. You may want to have a wide variation in well sizes in a first version of your mold to allow determination of the optimal sizes.
Injection needle production
Materials:
- Pipette Puller: Kopf Vertical Pipette Puller, Model 720
- Capillary Glass Tubing: Borosilicate glass capillaries 1.0 mm outer diameter (O.D) x 0.58 mm inner diameter (I.D.) x 100 mm long (L) Harvard Apparatus
- Bright-field Microscope with ruler
- Kimwipes
- Needle-holding Jar
First, set the pipette puller to an optimal heater voltage and solenoid current for your needs. We found that setting it to 14.9 units of heater voltage and the solenoid current to 4.7 amps DC regulated worked well for us. Insert the capillary glass tube into the machine and wait for the needle to drop. Wait a minute to allow the glass to cool; it can get really hot! This is where it gets to be less than perfect. The pipette puller is made to produce needle tips within 1-5 microns in diameter. In order for the needle to break through the chorion, it should be between 10-15 microns in diameter.
Hold the kimwipe gently in one hand and the needle in the other. With very light pressure, tap the needle onto the wipe. You should feel a very small snap. To make sure you’ve broken it to a satisfactory diameter, measure the diameter of the needle with a bright-field microscope fitted with a reticule.
**KEY point: We discard needles >15 microns as they result in high levels of death. Screen these for size under a microscope and ensure the outer tip diameter is low.
Note: The first few tries might result in a needle that is broken to 20 or more microns in diameter. This is expected! It takes some practice to get the right amount of pressure when tapping the needle on the Kimwipe.
Day of injections
Remove the barrier separating the fish and watch for spawning. I typically give the fish ~15 minutes, and if there is no sign of spawning, I try another day. If a female does spawn, note the time at which she first has eggs in her mouth. Embryos will be collected 30 minutes from this timepoint.
Prepare for injections. Mix the RNA/DNA/marker to be injected (as described in the Tol2/CRISPR sections), and backfill needles with a gel-loading pipet tip. I put ~1 µL into each of ~3-4 needles so that they can be rapidly switched out if one breaks.
Collect ~800 ml of water from the tank in which the spawning is occurring. Add methylene blue antifungal (final concentration, 1 mg/liter; Sigma) to the water, and then pipet 6 ml into each well of several 6-well plates. Plan to inject as many eggs as possible—usually 30-100 is possible—based on number laid, time until cell division, and speed of injection. Also use ≥10 embryos as uninjected controls. The survival rate among these embryos will indicate the viability of the brood and the quality of raising conditions. Any increased death above this rate can be attributed to the injections.
Injecting embryos
Fertilized eggs are collected ∼30 min after spawning, and injected during the single-cell stage.
Transgenesis mixture:
- Tol2-flanked transgene (225 ng/μL; final concentration, 75 ng/µL)
- Tol2 mRNA transcribed from pCS-TP (225 ng/μL; final concentration, 75 ng/µL)
- Texas Red-conjugated dextran* (final concentration, 0.5%; 3000 MW; Sigma) for visualization of injection. You can also use 0.05% phenol red, but TxRedDextran will enable you to see whether the injected solution is being taken up into the developing embryo, using a fluorescent dissecting scope.
We usually inject 2-3 pulses @ 2.8 ms, 40 psi (~1 nL per pulse).
Each injected egg was allowed to develop alone in a well of a 6-well culture dish (Greiner Bio-One).
This approach also allows us to track the progression of individual embryos. Try systematically varying injections and seeing whether survival/transgene uptake/etc is improved.
Care for injected fish
At ∼12 d postfertilization (dpf), just before the yolk is fully consumed, injected larvae were transferred to 1.5-L tanks, and then to 32-L tanks at ∼4-6 weeks of age.
At ~4-6 weeks post-fertilization, I take a fin clip, and PCR genotype (suggested protocol in appendix) for the presence of the transgene. In my experience, only those fish carrying transgene in their tail end up passing it through the germline.
Mate transgene carriers to wildtypes, and then check for transmission of transgene in G1 fish, using PCR or other method of verification. If it transmits, then congratulations, you’ve got a stable line! Now test whether it faithfully recapitulates expression in your targeted cell population, and see whether you get the phenotypes you’re looking for!
