線虫のニューロンに生殖細胞を再プログラムする RNAi と熱衝撃による転写因子の発現のアプリケーション

The overall goal of this procedure is to induce the conversion of germ cells to neurons in C.elegans. This method can help investigating the implication of biological processes, such as signaling pathways or epigenetics in the regulation of cellular fate reprogramming. The main advantage of the method described in this video is that in living animals, germ cells can be challenged by the overexpression of a fate-inducing transcription factor.

While in parallel, a genetic screen such as an RNAi screen can be performed in order to identify novel factors that are implicated in regulating cellular reprogramming. The implications of this technique extend toward regenerative therapy. Because this in vivo method could be used to study cellular reprogramming under different developmental and environmental conditions.

Visual demonstration of this method is critical, as the reprogramming steps are difficult to learn. Because the germ-cell-to-neuron conversion phenotype, upon depletion of lin-53, only happens in carefully-controlled conditions. For the RNAi plates, load six-centimeter plates with NGM agar containing IPTG and carbenicillin.

Let them dry for 24 to 48 hours at room temperature in the dark. Then transfer them to four degrees Celsius for up to 14 days. Next, streak selective plates containing carbenicillin and tetracycline with RNAi bacteria clones with L4440 plasmid carrying the lin-53 gene.

Use a three-phase streaking pattern, and be sure to plate an empty vector control. Then, grow the plates overnight at 37 degrees Celsius. The next day, pick at least three single colonies, and inoculate each of them into a separate culture tube with two milliliters of LB, supplemented with carbenicillin, but not tetracycline.

Plan to have three healthy cultures per condition. Next, grow the cultures overnight at 37 degrees Celsius until they reach an optical density at 600 nanometers of 0.6 to 0.8. Then, add 500 microliters of each bacterial culture to a six-centimeter NGM agar RNAi plate.

Incubate the plates overnight at room temperature in the dark. This protocol is optimized for BAT28-strain worms kept at 15 degrees Celsius, at which the transgenes are most stable. Details on the strain genetics are provided in the text.

Age synchronization of the worms is critical. Use the bleaching technique. Wash six-centimeter NGM agar plates containing adults and eggs using 800 microliters of M9.Then pellet the worms by centrifugation, and remove the supernatant.

Next, add 1/2 to one milliliter of bleaching solution to the worms. And while watching the worms under a stereoscope, shake the tube until the adult worms start to burst open, and then collect the eggs. Now, separate the waste from the eggs.

First centrifuge the tube, and dispose of the supernatant. Then wash the pellet of tissues with 800 microliters of M9 three times. Be sure to use centrifugation to pool the tissues between the washes.

Now, transfer the cleaned eggs to fresh NGM plates seeded with OP50 bacteria. Briefly check for successful isolation of embryos and their transfer to the NGM plates. Grow the animals at 15 degrees Celsius.

To achieve germ-cell-to-neuron conversion upon lin-53 depletion, the depletion needs to start in the parental generation. To deplete lin-53, subject L4 animals to RNAi. Manually transfer 50 L4 worms per replicate to an NGM RNAi plate without bacteria.

Use a platinum wire. L4 worms can be recognized by a white patch approximately halfway along their ventral sides. Now, incubate the worms at 15 degrees Celsius in the dark for about seven days.

The IPTG is light-sensitive. When the F1 progeny reach the L3 and L4 stages, separate them from the bigger, thicker parental animals. At this point, screen the F1 for the protruding vulva phenotype, which indicates that the RNAi against lin-53 has been successful.

The RNAi activity can also result in lethality, which increases above 15 degrees Celsius. To activate che-1, and induce the germ-cell-to-neuron conversion in the F1 progeny, heat shock the worms for 30 minutes at 37 degrees Celsius in the dark. Use a vented incubator, since it allows for a more efficient heat shock.

After the heat shock, incubate plates at 25 degrees Celsius overnight in the dark. It's important to avoid inducing overexpression of che-1 by heat shock before the animals reach mid L3 stage. This can lead to expression of che-1 protein in untargeted tissues.

The next day, set up a fluorescence light source and GFP filter to examine the animals for transgene-derived fluorescence in the mid-body area. To assess the phenotype penetrance, find the ratio of animals emitting a fluorescent signal. Typically, around 30%will show a discrete GFP signal.

By comparison, in animals carrying the empty vector RNAi, even a diffuse signal in the germline is not seen in more than 5%of the population. F1 animals that exhibit the protruding vulva phenotype illustrate successful application of lin-53 RNAi. In these animals, conversion of germ cells into ASE neuron-like cells upon heat-shock induction of che-1 is common.

Close examination reveals neuron-like projections that are typical of neurons from germline cells. By contrast, empty vector controls showed minimal expression of GFP, which was at most, diffuse. The germ cells in these animals lacked neuron-like features.

When the animals were too young at the time of che-1 overexpression induction, they would often display ectopic transgene expression in other regions of the body, such as the developing vulva. After watching this video, you should have a good understanding of how to carefully plan and perform the experiments using living worms in order to reprogram germ cells into neurons. The method described here in this video will allow you now to investigate the implication of a number of different factors in regulation of cellular reprogramming.

Once mastered, this procedure can be done in 10 days. While attempting the experiment, it's important to carefully stage the animals before the induction of reprogramming, and to keep the BAT28 strain at 15 degrees. Following this procedure, other methods like double RNAi can be performed in order to address additional questions like, What are the underlying mechanisms that play a role during cell fate conversions?