Question

1. Outline two experimental methods to test the hypothesis that marijuana alters a) gene expression and b) protein expression in mice brains. Explain also how you will know the specific identity of the genes and proteins that are altered. You will need to search the literature for biochemical techniques that would be appropriate.
2. Assume you find a gene that is upregulated in mice brains in response to marijuana. How would you determine exactly where in the mouse brain this gene is expressed and to what extent? In addition, how would you determine the subcellular localization of the protein corresponding to this gene? Briefly, outline five experiments to address these questions. You will need to search the literature for biochemical techniques that would be appropriate.

Answer 1

1. Outline two experimental methods to test the hypothesis that marijuana alters a) gene expression and b) protein expression in mice brains. Explain also how you will know the specific identity of the genes and proteins that are altered. You will need to search the literature for biochemical techniques that would be appropriate.

a) For analysis of gene expression the most widely used technique is RT-q-PCR (Reverse Transcription followed by Real Time PCR)

In order to check, if in a given tissue sample the transcription status of a set of genes is altered we will follow the following protocol:

1. Mouse will be used as model organism, let’s say we have taken 12 mice in total. These mice will be divided into 4 groups, group A, B, C and D; thus there will be 3 mice in each group, labeled as 1, 2 and 3.

Group A mice will be fed with marijuana: 1 gm per day

Group B mice will be fed with marijuana: 2 gm per day

Group C mice will be fed with marijuana: 4 gm per day

Group D mice will not be fed with marijuana and normal diet will be provided to them.

Except for the marijuana intake rest all the conditions in which these mice will be kept will remain exactly same. We want to ensure that the effects observed is due to marijuana intake only and not due to any other factors, thus we will try to keep the variables (like age, lineage, diet, environment and temperature etc) as less as possibly, preferably none.

1. We will monitor these mice for 3 months for which they will be given the specific amount of marijuana in their diet as mentioned above.
2. Mice A.1, B.1 C.1 and D.1 will be sacrificed on the 90^th day and their brain will be harvested separately.
3. A small section will be taken and minced.
4. Minced tissue will be dissolved in trizol and processed further by guanidinium thiocyanate-phenol-chloroform extraction method ( It is the standard method of extracting RNA from tissue or cell samples)
5. After taking the nanodrop reading, the RNA preparation will then be subjected to REVERSE TRANSCRITION, to prepare cDNA.
6. The cDNA will then be checked using primers for constitutive genes like RNA POL II, GAPDH etc. to ensure that proper cDNA formation.
7. Using Real Time PCR, then we will analyze the expression of our target genes.
8. Same protocol will be repeated with mice A.2, B.2, C.2 and D.2 after 180 days of marijuana intake; and mice A.3, B.3, C.3 and D.3 after 270 days of marijuana intake from experiment start date.

Now, we have expression of our targets in 4 different conditions, 3 different time point each. We will compare the expression of targets obtained for the different groups (A, B and C), subjected to marijuana for different time period with our control which is group D. This will give us a fair idea if marijuana is altering gene expression.

b) To explain that marijuana is altering protein expression profile of brain we can make use of techniques like WESTERN BLOTTING.

WESTERN BLOTTING: (Also called as Protein Immuno-Blotting) It is an analytical technique used to characterize presence of proteins in any sample. Here we actually co-incubate the target protein with specific antibody, which is tagged to reporter enzyme like alkaline phosphatase, HRP or a flurophore. That will help in indicating the presence of target protein.

The following protocol will be followed:

1. Tissue sample from the harvested brains or group A, B, C and D from different time points will be minced and homogenized. The cells will be lysed and the proteins will be solubilized using buffers and detergents (We ought to know the molecular wt. of our target proteins, whose expression we, want to analyze)
2. The proteins will then be loaded on a SDS gel (technique used here is SDS PAGE) and will be separated using electrophoresis. The proteins will be separated on the basis of their molecular weight.
3. In order to make the protein accessible to antibody we need to transfer it to an nitro-celluslose or PVDF membrane by electroblotting
4. In order to visualize the total protein that has been transferred to the membrane we may used stains like Ponceau Stain or Amido Black, this is to ensure that the total protein has been traced to the membrane.
5. The membrane tends to bind with all sorts of proteins. We want to avoid nonspecific interaction between antibody (protein) and the membrane, for this we will block the membrane using BSA (usually it is 2 - 5% Bovine Serum Albumin in TBS- Tris Buffered Saline with Tween- detergent- optional )
6. For detection we will co incubate the antibody specific against the protein of interest for 45 to 60 min in dark.
7. We will then add the substrate of the reporter enzyme, this convert the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane.
8. Now the specific bands for the target protein can be visualized.

Now, we have bands for the target proteins, the intensity and width will indicate the expression of our target protein in 4 different conditions, 3 different time point each. We will compare the expression of target protein obtained for the different groups (A, B and C), subjected to marijuana for different time period with our control which is group D. This will give us a fair idea if marijuana is altering protein expression.

If you do not want to undergo all this pain simply do a microarray for both gene expression and protein expression. It is a one step experimental process but very expensive. It has 4 different phases. (It is an extremely vast topic and its explanation will take a lot more time, but anyway if you are curious…… )

1. ISOLATION
2. HYBRIDIZATION
3. SCANING and QUANTITAION
4. ANALYSIS