Question

You are studying the part of the brain that mediates stress. You have treated some mice under stressed conditions and you also have control mice. You isolate the appropriate brain regions from both groups. Describe what you would do to identify changes in genes associated with acetylated lysine 9 on histone H3. What would you do to prepare the brain samples for analysis? Would technique would you use to purify genes associated with aceylated lysine 9 histone H3 and how does it work? How would you identify all the genes in the purified samples? What kind of data would you get in the end that would tell you what genes changed in their association with acetylated lysine 9 histone H3 in response to stress?

answer all please

no info needed

Answer 1

Here to perform this study we need both biochemical and global gene expression analysis.

First we need to grow mice in stressed and control conditions.

1. After the stress has been applied we need to isolate the brain region of interest in both experimented and control mice.
2. We need to perform a Chromatin immuno precipitation ChiP experiment to identify the gene of interest. First we will isolate the total chromatin from the brain tissue. This chromatin will be cross linked with formaldehyde to keep the histone DNA complexes intact. The crosslinked chromatin will be once purified using total chromatin isolation and will be proceed for fragmentation. DNA fragmentation can be performed using a sonicator which make DNA fragment of different sizes.
3. We need a primary antibody which can bind specifically to acetylated lysine of H Using this antibody we will incubate the fragmented chromatin and an immunoprecipitation will be performed to pull down the antibody H3 complexes. After the Salt washes we will be left with the specific DNA-H3-antibody complexes.
4. De -cross linking will be performed by treating with high salt washed where we will isolate and purify the DNA which is associated with the acetylated H3.
5. A DNA sequencing using next gen sequencer will be performed with the total chromatin which we immunoprecipitated from control ad stressed brain tissues.
6. This DNA sequencing will identify and annotate the differential gene expression j control and stress given mice.