We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.
Because Thermobia domestica is a basal insect, genetic studies on this species will illuminate mechanisms underlying the evolution of key innovations in insects, such as powered flight and metamorphoses. This protocol covers basic techniques from culture maintenance, to egg microinjection to implement Thermobia as a laboratory model species. This method can also be used for application of other genetic tools such as RNI mediated gene knockdown, and transgenesis in thermobia.
Maintain wild type and mutant populations in a large plastic container with regular artificial fish food, water in plastic cups with a ventilation hole on the top, a folded paper for hiding the insects and layered cotton for laying eggs. Keep all T.domestica cultures inside 37 degree Celsius incubators and set the relative humidity inside each container to 60 to 80%To prepare the egg collection colonies transfer about 20 male and 20 female adults to a middle sized container with food, a water supply, a folded paper, and a small piece of layered cotton for egg-laying. Set up several colonies to obtain a large number of staged embryos within a short time period.
On the day of injection replace the cotton inside the containers with new cotton. Eight hours later collect the eggs from the layered cotton by separating the layers using forceps. Align the eggs on the double-sided tape using a wet paint brush keeping a two millimeter distance between the eggs.
All eggs should be oriented so that the longitudinal axis of an egg faces the injection side. Gently press the eggs down with a paintbrush for firm holding during the injection. Load two microliters of the gRNA CAS9 solution into a glass injection capillary with a micro loader.
Make sure there are no air bubbles in the solution before the injection. If necessary tap the needle to remove the bubbles. optimize the shape of the needle tip by breaking it slightly with forceps to prevent clogging and to get better durability throughout a series of injections.
Insert the needle at the midpoint of the longitudinal axis of an egg and inject a small amount of the solution. Adjust the configuration of the electronic micro injector during injection, depending on the shape of the needle tip. Keep the injected eggs in an appropriately sized container with 60 to 80%relative humidity at 37 degrees Celsius.
Check the injected eggs periodically and discard damaged eggs to avoid fungal growth. If too much fungus is growing on an egg, clean the surface of the egg with 70%ethanol. Before hatching dip the glass slide with the injected eggs into talcum powder to coat the surface of the double-sided tape.
This will prevent the stacking of hatched nymphs. Transfer the powder coated glass slides to a middle-sized container with food, water, and a folded paper. Isolate G1 nymphs into 24 well plates with an aspirator or a paintbrush.
Maintain the supply of artificial regular fish food. Place the 24 well plates in a larger container with a water supply for a humidity of 60 to 80%Pinch and pull a cercus from a nymph or adult using forceps and collect them into a 0.2 milliliter tube containing 50 microliters of ethanol. Store the collected samples at negative 20 degrees Celsius for long-term storage.
Place sample tubes with the lids open on a thermal block for 15 minutes at 70 degrees Celsius to evaporate the ethanol. Injection of gRNA CAS9 ribonucleoprotein complex in embryos within the first eight hours after egg-laying results in indels at the gRNA targeted site. this causes biallelic mutations in some cells of the injected generation and mutant mosaic phenotypes are usually obtained.
When this protocol was used to inject a gRNA that was designed to target the white gene, 32.6%of G0 nymphs displayed a partial loss of pigmentation in their compound eyes and dorsal regions. Assessment of the germline transformation of G0 adults and mutated G1 individuals was done by genomic PCR followed by a hetero duplex mobility assay. In G1 samples, differential band patterns between wild type and mutated samples are clearly distinguishable.
Genome editing in Thermobia provides a solid approach to analyze genetic mechanisms underlying premature traits of insects. It helps to explore the secrets behind their outstanding success and ours.
