This protocol enables a researcher to identify mutant sea anemones during early embryogenesis so that post-embryonic developmental phenotype can be analyzed. The main advantage of this technique is that it can be used to genotype individual sea anemones early in ontogeny without sacrificing the life of the animal. Demonstrating this procedure will be Miguel Silva, a graduate student from my laboratory.
On the day before spawning induction, place the anemone nematostella vectensis in a temperature-and light-controlled incubator, programming the incubator so that the animals are exposed to eight hours of light at 25 degrees Celsius. On the following day, remove the animals from the incubator and leave them on a bench top at room temperature with the light on to allow spawning, which will occur within the following one and a half to two hours. Use a transfer pipette who's tip is cut to enlarge the opening to place egg packages from the female container into a sperm-containing male container and leave them in the male container for at least 15 minutes to allow for fertilization.
After fertilization, to dejelly the egg packages, place them in seawater containing 3%cysteine on a glass Petri dish and gently agitate on a shaker for 12 minutes. Then break up clumps with a plastic pipette and continue to agitate for another two to three minutes until completely dejellied. Keep the fertilized eggs on the same dish at 16 degrees Celsius or at room temperature.
On the next day, prepare DNA extraction buffer as described in the manuscript, mix well by vortexing, and aliquot it into PCR tubes by adding 20 microliters to each tube. Dissolve 1%agarose in seawater, pour it into a Petri dish to cover the bottom, and cool on a bench top to make a gel bed. Cover the gel bed with fresh seawater.
Transfer 24-hour post-fertilization embryos using a pipette into the agarose gel bed-containing Petri dish. Insert a tungsten needle into a need holder and sterilize by dipping its tip in alcohol and placing it in flame to burn off the alcohol. Under a dissecting microscope, make a depression on the agarose by using the tungsten needle to remove a piece of surface agarose about size of an embryo to be manipulated.
Then, using the tungsten needle, place the embryo into the depression with its lateral side facing down in order to restrict the movement of the embryo for microsurgery. In order to perform successful surgery, it is critical to restrict the movement of the embryo. With the tungsten needle, surgically remove a piece of aboral tissue along the oral aboral axis, located opposite to the oral blastoporal opening.
With a P20 pipette, transfer the isolated aboral tissue to a PCR tube containing 20 microliters of DNA extraction buffer. Transfer the post-surgery embryo into a well of a 24-well plate containing at least 500 microliters of fresh seawater. And place the plate containing post-surgery embryos in an incubator at 16 degrees Celsius until genotyping is completed.
To extract genomic DNA from single embryos, first briefly spin down the PCR tubes containing the DNA extraction buffer and isolated embryonic tissues using a mini centrifuge. Then incubate the tubes at 55 degrees Celsius for three hours while vortexing for 30 second every 30 minutes to ensure breakup of cell clumps and enhance cell lysis. After inactivating proteinase K at 95 degrees Celsius for four minutes, set up a PCR reaction using extracted genomic DNA as a template to amplify the locus of interest.
Design desired primers as described in the manuscript. Set up a 20 microliter PCR reaction. After completed PCR, run agarose gel electrophoresis to determine the size and presence or absence of PCR products, making sure to adjust the condition of agarose gel electrophoresis depending on the expected size of PCR products.
Use PCR results regarding the size and presence or absence or PCR products to assign a genotype to each post-surgical embryo and then sort embryos according to their genotype. The nematostella genome has a single locus that encodes a precursor protein for the neuropeptide, GLWamide. Three knockout mutant alleles at the locus have been previously reported.
PCR results from randomly sampled embryos among the progeny of an F1 cross between one heterozygous female carrying an A knockout allele and heterozygous males carrying a C knockout allele show different genotypes. Embryos one and two show a single PCR band corresponding the expected size of A knockout allele. Embryos three and six show two PCR bands with the expected sizes for knockout alleles A and C.Embryos four, seven, and eight show a single PCR band corresponding to the expected size of C knockout allele.
Embryo 5 shows no bands, suggesting the lack of primer binding. To rule out the possibility of genomic DNA extraction failure, another PCR was run using a reverse primer that can bind to the wild type sequence. Where a PCR product of an expected size was detected, genomic DNA extraction was successful.
Whereas no product suggested a failure in extraction. Although this protocol is designed for sea anemone embryos, it sure be possible to apply this method to other cnidarians, such as corals and jellyfish, where genomic information and embryos are accessible. Following this procedure, the identified mutants are used to assess developmental phenotype.
For instance, histological techniques such as immunostaining can be used to assess defects in morphology, while in situ hybridization, realtime quantitative RT-PCR, and RNA-seq can be used to assess defects in gene expression. Also, live imaging can be used to assess defects in behavior during post-embryonic development, such as in larvae or in early polyps.
