Are there other experiments that must be performed in order to get Dicer II ready for the gene knock-out experiments, for example the in-vivo fruit flies experiment mentioned?
In order to do a knockout experiment, we would have to either remove the gene from the fruit fly genome (using CRISPR, which takes time to prepare and test) or maybe use RNAi to prevent the formation of the DICER II protein in drosophila eggs. Either way, a bit of preparation is needed to make sure these methods inhibit only DICER II and don’t block other DNA damage response pathways.
Are there other experiments that need to performed before Dicer II is ready for gene knock out experiment like the in-vivo fruit fly experiment you mentioned?
The field of DNA damage response is still determining the molecular mechanisms that cells use. The first step in learning about these processes is learning which genes are involved, which is how our research plays into it! Now that we know DICER II may be involved in DDR, we can begin to narrow down the possibilities for mechanisms. In particular, we are still learning a lot about RNAi which is involved in this pathway.
In this experiment, there were only DSB inducted by the hydroxy urea treatment! Its possible that DICER II is involved in treating single-stranded breaks, but more research would be needed. My guess is that DICER II creates RNAi which are involved in a pathway that activates non-homologous or micro homology end joining. These are not involved in single-stranded breaks so DICER II may not respond to these but more testing is necessary.
This is a really interesting question because the small RNAi pieces DICER II creates could technically regulate anything! It is possible that the effects of DICER II are more broad than just DNA repair. Perhaps these molecules are used for signal transduction or to stabilize DNA during molecular DNA handling. RNAi is not fully understood yet, so a knockout experiment in cells or in vivo drosophila would begin to show us how DICER II affects the cell. (For instance, if double knockout of the gene causes cell death, the protein might be important for many different pathways. If fruit flies survive the double knockout but age quickly, DICER II may be involved in aging and longevity pathways related to DNA damage.)
The other lanes contained a positive control gene assay that we knew would increase in expression after DNA damage (Rad51) and a loading control that would not increase in expression (Rpp0). For both of these gene assays, we included samples treated with reverse transcriptase and samples without, which served as a negative control where no bands were expected. Each gene assay also included a sample treated HU to induce DNA damage and a sample that was untreated so we could compare expressions.
Good Job! Is there any particular reason why you mentioned choosing fruit flies to knock genes to expose the mechanism of Dicer2 as oppose to other organisms such as C. elegans?
curesymposium.org 
Are there other experiments that must be performed in order to get Dicer II ready for the gene knock-out experiments, for example the in-vivo fruit flies experiment mentioned?
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In order to do a knockout experiment, we would have to either remove the gene from the fruit fly genome (using CRISPR, which takes time to prepare and test) or maybe use RNAi to prevent the formation of the DICER II protein in drosophila eggs. Either way, a bit of preparation is needed to make sure these methods inhibit only DICER II and don’t block other DNA damage response pathways.
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Are there other experiments that need to performed before Dicer II is ready for gene knock out experiment like the in-vivo fruit fly experiment you mentioned?
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How does your research play into other experiments done in this field?
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The field of DNA damage response is still determining the molecular mechanisms that cells use. The first step in learning about these processes is learning which genes are involved, which is how our research plays into it! Now that we know DICER II may be involved in DDR, we can begin to narrow down the possibilities for mechanisms. In particular, we are still learning a lot about RNAi which is involved in this pathway.
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Is a double stranded break needed in order to see an increase in expression?
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In this experiment, there were only DSB inducted by the hydroxy urea treatment! Its possible that DICER II is involved in treating single-stranded breaks, but more research would be needed. My guess is that DICER II creates RNAi which are involved in a pathway that activates non-homologous or micro homology end joining. These are not involved in single-stranded breaks so DICER II may not respond to these but more testing is necessary.
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What other types of pathways could possibly be involved? How would results from the in-vivo fruit fly experiment support this?
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This is a really interesting question because the small RNAi pieces DICER II creates could technically regulate anything! It is possible that the effects of DICER II are more broad than just DNA repair. Perhaps these molecules are used for signal transduction or to stabilize DNA during molecular DNA handling. RNAi is not fully understood yet, so a knockout experiment in cells or in vivo drosophila would begin to show us how DICER II affects the cell. (For instance, if double knockout of the gene causes cell death, the protein might be important for many different pathways. If fruit flies survive the double knockout but age quickly, DICER II may be involved in aging and longevity pathways related to DNA damage.)
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What did the other lanes contain on the gel and were they relevant in the experiment?
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What did the other lanes of the PCR contain and was the findings relevant to the resuts?
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The other lanes contained a positive control gene assay that we knew would increase in expression after DNA damage (Rad51) and a loading control that would not increase in expression (Rpp0). For both of these gene assays, we included samples treated with reverse transcriptase and samples without, which served as a negative control where no bands were expected. Each gene assay also included a sample treated HU to induce DNA damage and a sample that was untreated so we could compare expressions.
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How does this study of Dicer II apply to the study of DNA in humans, what is the applicability?
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Good Job! Is there any particular reason why you mentioned choosing fruit flies to knock genes to expose the mechanism of Dicer2 as oppose to other organisms such as C. elegans?
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