The overall goal of this methodology is to derive stable tetraploid strains from diploids of any genotype or karyotype in order to study the role or consequences of whole genome polyploidization in biological processes. This method can help study the role of whole genome polyploidization on gene dosage, biological scaling, extracellular signaling, genome instability, development of resistance to drugs, adaptation to stress, and mechanisms of speciation. The main advantage of this technique is that it's efficient, versatile, simple, as well as the only available technique in multicellular organisms to generate stable, fertile tetraploids from any diploid strain.
I first had the idea of this method when I was analyzing the chromosome segregation phenotype of rec-8 mutants in spermatocytes and oocytes. Rec-8 mutants generate diploid sperm and oocytes indicating that reducing the rec-8 gene expression level could result in production of tetraploid progeny at fertilization. Demonstrating this procedure will be Katherine Rivera Gomez, a graduate student of my laboratory.
To induce rec-8 double-stranded RNA expression in Escherichia coli, prepare NGM agar plates with one millimolar IPTG and 100 micrograms per milliliter of ampicillin. Store these plates in the dark at four degrees Celsius for up to four weeks. Now prepare the bacteria carrying the appropriate rec-8 clone.
Streak them onto the plates containing the appropriate antibiotics and incubate the bacteria overnight at 37 degrees Celsius. The next day, inoculate single colonies from the plates into four milliliters of LB with the same antibiotic concentration and grow the bacteria overnight at 37 degrees Celsius with agitation. The following day, induce production of double-stranded RNA by adding IPTG until the final concentration is one millimolar IPTG.
Then continue the incubation for 40 minutes. Next, seed the prepared NGM IPTG plates. Add 100 to 200 microliters of culture to the plates and incubate them at room temperature in the dark overnight.
The next morning, place three to four young L4-staged hermaphrodites onto the rec-8 bacteria plates and let them grow up at 15 degrees Celsius in the dark. About three days later, when the F1 hermaphrodites are at the L3 to L4 larval stage, start making more rec-8 RNAi bacteria plates. After about four days on the rec-8 bacteria plates, transfer 20 of the F1 L4 young adult hermaphrodites onto the freshly induced rec-8 RNAi plates and allow them to self-fertilize.
It is also possible to include four to six untreated males of the same strain. Later, check for F2 progeny with the long phenotype. They are overall larger than the wild type and their bodies make an extra turn as they move forward generating sinusoidal body bending waves from head to tail.
Transfer these putative tetraploids onto regular OP50 or HB101 E.coli bacteria. Screen the plate for F3 progeny as well. However, these F3 cannot be considered independent strains because they may be siblings from the same F2 mother.
Let the collected long worms self-fertilize to propagate. Continue transferring three to six long animals from each generation to a new plate until only long progeny are sired. This may take a few generations as long worms often are sterile and do not yield progeny.
Tetraploid strains have 12 chromosome pairs which can be validated by counting the DAPI stained bodies in unfertilized oocytes. To do so, drop five to 10 microliters of M9 buffer onto a slide and transfer six to 10 putative tetraploids into the drop. Under a dissecting microscope, carefully soak up most of the M9 into a lint-free cleaning tissue.
Then drop 10 microliters of 90%ethanol onto the worms and watch the ethanol evaporate completely. As soon as the evaporation finishes, repeat the process of adding ethanol and watching it evaporate. In total, add 10 microliters in four applications.
After the last drop evaporates, add six microliters of DAPI at two nanograms per microliter or a similar stain. For long-term storage of the slides, mount the worms in a commercially available or homemade anti fade. Then add a coverslip and seal the edges with nail polish.
After the nail polish has dried, the slides can be scored. Use a fluorescent microscope and 100X magnification. First find the most mature unfertilized oocyte which is immediately adjacent to the spermatheca and has not yet entered either the spermatheca or the uterus.
The DAPI bodies here are presumably single chromosome pairs. Next, focus on the nucleus of the oocyte and use the microscope's fine focus to slowly scan it from top to bottom while counting the DAPI bodies. Then recount the DAPI bodies in the same nucleus by moving the focus from bottom to top.
Wild type oocytes have six DAPI bodies on average. The presence of 12 DAPI bodies indicates that the animals in this strain are either partial or complete tetraploids. Analyze at least 10 animals per strain.
Often chromosome pairs will be very close or touching so the number of DAPI bodies is often smaller than the actual number of chromosome pairs. Multiple tetraploid strains were generated by feeding C.elegans bacteria expressing rec-8 double-stranded RNA. Tetraploids can arise by self-fertilizing hermaphrodites for two to three generations or by crossing the first generation of hermaphrodites with untreated males of the same genotype.
In the latter scenario, the males in the cross are exposed to the rec-8 RNAi bacteria from the L4 stage onwards during mating. Tetraploid strains were confirmed by screening for the presence of 12 chromosome pairs found in the oocytes of the tetraploid hermaphrodites compared to six chromosome pairs found in the oocytes of diploid hermaphrodites. Two types of tetraploid hermaphrodites were identified.
One sired males at similar frequencies to diploid hermaphrodites and the other sired males at a much higher frequency. The former is tetraploid for all chromosomes and the latter is tetraploid for all autosomes but is triploid for the sex chromosome. Tetraploid strains grow slower and produce smaller broods.
Superficial inspection of the tetraploid strains has not revealed sufficiently elevated numbers of aneuploid oocytes nor abnormal oocyte or spermatocyte divisions to account for the observed reduction in brood size. After its development, this technique has paved the way for researchers in the field of developmental cancer and evolutionary biology to explore the role of whole genome polyploidization on a great variety of biological functions. When using this technique, it is important to remember to always use fresh NGM IPTG plates to induce bacterial expression of double-stranded RNA.
If everything goes well, tetraploids can be produced in less than three weeks'time.
