This study investigates the use of genetically engineered bacteria to facilitate neuronal differentiation in nematodes through RNA interference. By downregulating a chromatin-regulating factor, the research demonstrates a novel approach to reprogram germ cells into neurons.
Take genetically engineered bacteria on a nematode growth medium.
The bacteria carry a plasmid with a nematode chromatin-regulating factor gene.
The medium contains an inducer that triggers the expression of a transgenic RNA polymerase from the bacterial genome that transcribes the chromatin-regulating factor gene into a dsRNA.
Add transgenic nematode larvae carrying a neuron fate-inducing transcription factor gene under a heat-shock promoter.
Upon bacterial consumption, the ingested dsRNA generates siRNA that downregulates the chromatin-regulating factor expression, facilitating cell fate reprogramming.
Through the parents' germ cells, the siRNA is passed onto the progeny.
Downregulation of the chromatin-regulating factor expression in the progeny germ cells, indicated by a protruding vulva phenotype, allows their reprogramming.
Apply heat shock to the progeny, activating the heat-shock promoter and expressing the neuron fate-inducing transcription factor that triggers neuronal differentiation, transforming germ cells into neurons.
The neurons express a transgenic fluorescent reporter protein, facilitating microscopic visualization.
Manually transfer 50 L4 worms per replicate to an NGM RNAi plate. 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 G1 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 is important to avoid inducing overexpression of Q1 by heat-shock before the animals reach mid-L3 stage. This can lead to expression of Q1 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 midbody area.
繁體中文
