Jul
31

An Uninspired Title for My July Post

I’ve spent a hectic July in and out of the tissue culture room. During this time, I switched out my old wild-type rat thyroid hormone receptor with human TR and had to rework the concentrations I was using in my transfection procedure. Nevertheless, it was generally a painless process, whose only impediment was my hatred of long decimals that cannot be divided without a calculator. The results of two previous transfections I discarded, as they had utilized the rat TR and I wished to fully switch over to its human counterpart for the remainder of the assay.

I completed my requisite transfections last week and am spending the remainder of my time staking out the microscope room. My only complaint is that the microscope room is cold. (I am wearing sweaters during mid-summer because of its refrigerator-like quality.) During my time spent on the microscope, I spotted a cell that looked like a frog and a cell that looked like Edvard Munch’s The Scream. Regrettably, I have not found any cells that resemble A) dragons or B) myself, so I consider it a loss.

In any case, I am halfway through scoring my plethora of slides. My goal is to collect all of the raw data before the end of the summer session, and then analyze the localization of my mutant relative to wild-type TRa1 over the short break before fall semester begins.

Comments

  1. Kristin Passero says:

    Hi Chen!

    We call it a “semi-quantitative” scoring method. We designate a region of interest both in the nucleus and in the cytoplasm of a given cell that is expressing the receptor of interest. The Nikon program records the fluorescence of these two areas and we export the data into a separate file for further analysis.

    There isn’t a true difference in localization between rat and human TR in cells. In fact, our lab used to use only rat TR in its experiments! (We recently switched over). The nuclear import and export signals are highly conserved among TRs and the few mutations between rat and human TR were not in any regions that would have compromised the research we do on nucleocytoplasmic shuttling. I simply revamped my transfections using only human TRa1 because I didn’t want half of my trials to use rat TR and the other half to use human.

  2. Hi Kristin,

    I totally get the feeling that the microscope room is freezing! I always need to bring a jacket with me when I work there. When you mentioned scoring your slides, I’m wondering if you could briefly elaborate on how you do this? Is is something qualitative or quantitative? I’m also curious if there’s any difference between the localization of rat TRa1 and human TRa1 in cells?

    Good luck!

    Best,
    Chen

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