Cell Culture Success and Preparing for Toronto

In the last two weeks of June (yes, this is a retroactive post), I finally had some success with cell culture! One of the more recent tubes of PC12s that I thawed looked great after letting them be for a couple weeks. I was able to do two trials of an experiment with our new plasmids! At the beginning of this summer, Dr. Hinton had new plasmids cloned with more exact cuts for the truncations of MK-STYX. We also now have both GFP and mCherry (two different fluorescent tags for viewing under a fluorescent microscope) versions for both F1 (the active mutant of MK-STYX, which has phosphatase activity) and the truncated versions of the two domains of MK-STYX, CH2 and DSP. My project looks at the effects of the two domains of MK-STYX individually in neuronal cells, so these new constructs are very relevant. Unfortunately, when viewed under the microscope, we barely see any mCherry for the cells transfected with CH2-mCherry or DSP-mCherry, so that’s something I’m going to have to figure out when I get back from Canada.

Speaking of Canada, I’m going to be in Toronto in Anne-Claude Gingras’ lab in the Lunefeld-Tanenbaum Research Institute at Mount Sinai Hospital for three weeks in July! Her lab focuses on proteomics and mass spectrometry and does very exciting research, so I know it’ll be a great experience. In the weeks leading up to leaving for Canada, I started reading up on mass spectrometry and BioID, a process that allows you to tag and identify proteins that interact with or near a target protein.

Comments

  1. Nice job getting the cells to culture! I know you were having trouble with that in your last blog post. Is there any way for you to upregulate mCherry expression by using different promoters in your plasmid?

  2. kmreed01 says:

    Thanks! While I’m having issues seeing the mCherry constructs in PC12 cells, we have been able to see expression in HEK293s. So it’s probably an issue with transfection rather than with the plasmid itself, and next I’ll look to optimize transfection for these cells.