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The overall goal of this procedure is to introduce mutations into specific genes in the cave fish Astyanax mexicanus. This method can help answer key questions in the evolutionary biology field about the genetic basis of the evolution of cave traits. The main advantage of this technique is that the effect of loss of the specific gene can now be tested in Astyanax mexicanus.

After designing and assembling transcription activator-like effector nucleases, or TALENs, according to the text protocol, use two microliters of Sac1 to digest four micrograms of sequence verified template at 37 degrees Celsius for two hours. Run two microliters of the Sac1 digested plasmid on a one point five percent agarose gel and check for a single band to verify digestion. To carry out T three mRNA production, use zero point five micrograms of linearized template to set up half reactions following a standard protocol.

Incubate at 37 degrees Celsius for two hours. Then add zero point for microliters of DNase and incubate at 37 degrees Celsius for 15 minutes. After purifying the mRNA, to check the quality, use a product that eliminates RNase contamination to clean the gel apparatus.

Then, prepare a one point two percent gel. Mix one microliter of the mRNA, four microliters of nuclease-free water, and five microliters of glyoxal loading dye. Incubate the samples at 50 degrees Celsius for 30 minutes.

Then briefly centrifuge the tubes, then place them on ice before running the gel. After checking the concentration the mRNA, prepare aliquots before storing at minus 80 degrees Celsius. Refer to the text protocol for additional details.

After preparing injection needles and plates according to the text protocol, induce spawning in A.mexicanus by overfeeding fish for three to four days prior to breeding and place the fish into fresh water. Raise the water temperature two degrees Fahrenheit to approximately 76 degrees Celsius. Every 15 minutes, check for surface fish eggs in the dark and transfer them into glass bowls to prevent them from sticking to the surface.

Hundreds of eggs can be obtained from a single pair of surface fish. Under a microscope, sort the isolated eggs and transfer those at the one cell stage to fresh system water. Load the diluted mRNA into the back of the needle and detach the needle to a microinjector.

Then, using forceps, break the needle. To calibrate the needle, inject 10 times and collect the resulting drop in a microcapillary. The drop should fill a 10 by 100 disposable one microliter, 32 millimeter microcapillary to zero point five millimeters per one point five nanoliter injection.

Now, insert the needle into the single cell and inject the mRNA. Collect the injected embryos in glass bowls and incubate them at 23 to 25 degrees Celsius. Remove dead embryos regularly for the first few days following injection.

Record the number of dead and deformed embryos from uninjected control and injected plates. Refer to the text protocol for additional details. After euthanizing fish according to your institutions IACUC protocol, to extract the DNA, collect the embryos into 100 microliters of 50 millimolar of sodium hydroxide and incubate at 95 degree Celsius for 30 minutes.

Then cool to four degrees Celsius. Add a one tenth volume of one molar tris HCL ph 8 to the cooled embryos. Then carry out PCR using primers, for example for the oculocutaneous albinism two or OCA2 gene.

For individual embryos, one microliter of DNA is sufficient for the PCR reaction. To genotype the OCA2 locus, use the restriction enzyme DSR1 to digest the resulting PCR product by adding zero point five microliters of enzyme directly to twelve point five microliters of the completed PCR reaction. After incubating at 65 degrees Celsius for two hours, run the reaction and a sample of undigested PCR product on a one point five percent agarose gel.

Carry out additional analyses according to the text protocol. TALEN injections can result in double stranded DNA breaks that can be repaired by non-homologous end joining but which can also introduce insertions or deletions, or INDELs. Using PCR end restriction digestion to target the TALEN spacer region INDELs can be identified by the presence of a restriction enzyme resistant band.

TALEN injections can produce biallelic gene mutations in A.mexicanus and thus some phenotypes can be assessed in founder fish. For example, OCA2, the gene hypothesized to be responsible for albinism in cave fish, was evaluated. As shown here, albino patches in OCA2 TALEN injected fish were identified that were not present in uninjected fish.

In this gel image, 306 base pair PCR products from exon nine of OCA2 of A.mexicanus, were examined for loss of the restriction enzyme site in pools of 10 F1 fish from an injected founder fish. The amplicon from a control embryo was digested while a portion of the amplicon was resistant to restriction digest in F1 embryos from injected fish. In addition, the restriction digest resistant bands have been shown to contain INDELs.

Once mastered, these injections can be done in less than a few hours if they're performed properly. While attempting this procedure, it's important to remember to work carefully with RNA to prevent degradation and to inject during the one cell stage. Following this procedure, phenotypic analysis can be performed in order to answer additional questions such as if a particular gene underlies a key fish phenotype when mutated in surface fish.

This technique will pave the way for researchers in the field of evolutionary biology to characterize the role of candidate genes in the evolution of the cave fish Astyanax mexicanus. After watching this video, you should have a good understanding of how to inject Astyanax mexicanus embryos with TALEN mRNA.