Wednesday, June 2, 2010

Our first GFP-RFP strain

On aspect of the lab that I haven't wrote about much is our Scan^R microscope. The problem is that I planned to explain it with a lot of details, which meant that this post was being postponed everytime.

In the mean time, however, we have been using it quite a lot. For our purposes, we need strains that contain the right fluorescent markers. We relay on the use of  proteins, such as the Green Fluorescent Proteins (GFP) that are inherently fluorescent. This means that when the protein is expressed (made by the cells) we can measure it.

To setup experiments we need to make sure the cells express these proteins in response to the conditions we want to test. As our first attempt in that direction we combined strains expressing GFP attached to different native proteins (one per strain) with a strain that Avital built that expresses a protein called mCherry (a variant of Red Fluorescent Protein or RFP) in a constitutive way. This means that all cells are marked in red, which helps focusing on them and also defining the area of the cell.

We had some set backs with getting the mCherry strain to work, but after some detective work by Avital, she overcame the technical issues. Last week Avital managed to get her mCherry strains to mate with the GFP strains. And next week we are supposed to get the final product of haploid cells with the two markers. In the mean time she took images of the diploid cells, and they look good.

In red:

In Green:

And an image made by overlaying these two images:

You can see that (almost) all cells are red (with varying degrees). Some cells have green in the nucleus (sharp points in the middle of the cells), suggesting that this protein is nuclear one and has variable level of expression under the conditions we measured.

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