How to test neuronal silencing in drosophila pupae?

04 January 2023 1 7K Report

I need to silence dopaminergic neurons during the second neurodevelopmental stage of a Drosophila, i.e., during the pupal stage when most of the neurons that are required for the adult fly brain starts its formation. I have two tools with me: GtACR1 and Kir2.1, Tub GAL 80ts. But I am unable to monitor the neuronal silencing that occurs during that particular time of the fly life span. So, it would be great if I can get help about how to test whether my targeted neurons are actually silenced for the desirable time point during development.

Rodolfo Bolaños Barrera

Neuronal silencing, also known as gene knockdown, is a technique used to inactivate the expression of specific genes in order to study their function. In Drosophila melanogaster, one of the most common techniques for neuronal silencing is the use of RNA interference (RNAi) through the injection of double-stranded RNA (dsRNA) into pupae. Here is a general overview of how to test neuronal silencing in Drosophila pupae:

Obtain the dsRNA: The first step is to obtain the dsRNA that corresponds to the gene of interest. This can be done by synthesizing dsRNA in vitro or by using commercially available dsRNA.

Inject the dsRNA: The dsRNA is then injected into the pupae using a microinjection system. The dsRNA is usually injected into the body cavity, but it can also be targeted to specific cells or tissues by injecting it into the developing nervous system.

Monitor the phenotype: Once the dsRNA has been injected, the pupae are allowed to develop into adults. The phenotype of the adults can then be monitored to determine the effects of the neuronal silencing on the specific gene of interest.

Confirm the silencing: To confirm that the gene of interest has been silenced, it can be measured the expression of the gene by quantitative PCR or by Western Blot.

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