Cave animals provide a compelling system for investigating diverse morphological, developmental, and behavioral traits. But one of the limiting factors in establishing this model system is a lack of genetic tools that would allow us to manipulate gene function. Here we demonstrate three different approaches to manipulate gene function in the Mexican cave fish, Astyanax mexicanus.
These tools will enable the investigation of the genes underlying natural variations between surface fish and cave fish and how they relate to phenotypes. Bethany Stahl, a talented postdoc in my laboratory will demonstrate this procedure. Before beginning the procedure, fill a 100 milliliter Petri dish with warm 3%agarose dissolved in fish system water and carefully place an egg injection mold into the agarose at a 45 degree angle, before slowly lowering the mold into the agarose to avoid trapping air under the mold.
When the agarose has solidified, carefully remove the mold then store the plate at four degrees Celsius for up to one week. Then pull needles from borosilicate glass capillaries and an electrode needle puller according to the manufacturer's guidelines. On the day of the injection, use a glass pipette to transfer single cell eggs into the injection plate, filling up to five rows of the injection plate with 30 to 40 eggs per row.
Keep the eggs hydrated with a small amount of fish system water. Thaw Morpholino on ice and prepare the injection solution so that 400 picograms of Morpholino is injected per egg by using a long Microloader pipette tip or adding a two to four microliter bolus to the end. When all of the needles have been loaded, use forceps to trim the excess length from the injection needle tips and mount the first needle into a micromanipulator connected to a picoliter microinjector.
Then, place the injection dish under a dissecting microscope and use the micromanipulator to penetrate each egg with the needle before injecting one nanoliter of Morpholino injection solution directly into the yolk. For CRISPR injection, mix 25 picograms of guide RNA with 300 picograms of CRISPR-associated protein 9 messenger RNA and inject two nanoliters of the resulting RNA solution into each embryo as demonstrated. For Tol2 transposase and Tol2 flanked plasmid injection, combine 25 nanograms per microliter of Tol2 messenger RNA with 25 nanograms per microliter of Tol2 plasmid construct and phenol red in RNase-free water.
Then inject one nanoliter of solution into each embryo as just demonstrated. To screen the Morpholino-injected animals at four days post injection, depending on the gene of interest, visualize the fish larvae under a stereo microscope to observe the phenotype of the injected animals or video record to measure behavior. To screen for CRISPR indels, transfer CRISPR-injected embryos into PCR tubes and extract the DNA according to standard DNA extraction protocols for polymerase chain reaction or PCR analysis.
To assess for mutagenesis, run five microliters of the PCR product on a 3%agarose gel at 70 volts for three hours. Wild-type, non-mutagenized DNA will result in a distinct band, while mutant DNA will result in a smeary band. Cave A mexicanus injected with a control Morpholino exhibits significantly more locomotor activity and reduced sleep over a 24-hour period, compared to surface fish injected with a scrambled Morpholino.
The injection of the hypocretin orexin Morpholino has little effect on sleep in surface fish compared to control-injected fish. In contrast, hypocretin orexin knockdown via Morpholino injection has a significant effect on sleep in cave-dwelling fish, providing a direct link between hypocretin orexin expression and sleep loss. CRPSR-mediated mutagenesis of the albinism gene OCA2 in surface fish results in some individuals homozygous for the wild-type OCA2 positive allele, and is associated with harboring pigmentation, whereas individuals that have two copies of the CRISPR-mutated OCA2 negative allele are albino, providing a direct link between the mutant OCA2 gene and albinism in cave fish.
Select F0's positive for the Tol2 transmitted transgene to back cross to the wild type to generate stable F1 lines. This enables live calcium imaging for uncovering differences in neuronal activity, mediating behavioral changes in the cave environment, laying the groundwork for the expression of many additional transgenes to characterize and manipulate gene function in A mexicanus. The most important things to remember are to load the eggs tightly onto the plate, to trim the needle properly, and to optimize the microinjection.
After a successful genetic knockdown, transgene integration, or CRISPR-mediated gene editing, these fish can be used for developmental, behavioral, and brain activity analyses.
