Shockingly Transfected

Hey Hey!  Well, today marks the midpoint of my trip.  In the two weeks I have been here I feel like I have done/seen so much.  It is comforting to know that I have yet another two weeks to do even more!

This week, in the lab, I have been working with Dr. Mori who is a senior scientist at NIRS.  He has been wonderfully helpful and has made a great attempt at explaining things to me in English.  First, he let me practice cell culturing (i.e. washing, trypsinizing, and splitting) with a human colon cancer cell line known as HCT 116.  Later he showed me how to build a DNA vector from E.Coli to be used for transient (as opposed to stable) transfection of a gene.  Apparently, transient transfection allows for a higher quantity of gene expression to occur for a brief period of time (http://en.wikipedia.org/wiki/Transfection).  Today we performed electroporation, a type of transfection process in which foreign DNA is introduced into a eukaryotic cell via formation of transient pores in the cell membrane by high voltage pulses.  In the lab,  cells are placed in suspension in an appropriate buffer (we used K-PBS) and into an electroporation cuvette.  This cuvette is then connected to a power supply and the cells are essentially “shocked” by a high-voltage (today we used 300V & 960µF capacitance) electrical pulse.  After this electrocution 30-50% of the cells actually die and the remainder are what will give us expression of the desired gene.  In this case, we transfected HCT116 cells with a plasmid containing mKate2, a highly fluorescent monomeric fusion protein (http://www.evrogen.com/products/mKate2/mKate2.shtml).

This model/method is convenient and ideal for many of the types of experiments that are performed here at NIRS; one component of the cancer & radiation research is DNA Repair.  When DNA strand breaks occur due to some toxic insult such as heavy ion radiation (i.e. C or Fe), there are many expression factors that appear at the sight of DNA repair.  Certain fusion proteins such as mKate2 are able to fuse to those expression factors and serve as great indicators of that DNA repair.  So there are many implications in the building of these cell lines which contain fluorescent fusion proteins.  Did I mention that it makes the cells pretty?  The fluorescence of the cells is quite attractive to the eyes and apparently, depending on the protein(s) being used, one can stain up to five different colors at once!  Dr. Mori said it looks like a rainbow.  hehe.

In addition to the lab stuff I have gotten to go to the beach, to a Buddhist temple and the big Buddha statue in a town called Kamakura, and I’ve been to a very sweaty and crowded Japanese summer festival!  Plus, I have been exploring the cute little town of Chiba on my runs and the parks are quite pleasant.  That is only a brief summary of the wonderful things I have been experiencing and I’ll try to inform you of more the next chance I get!  Sayonara for now!

-Petra

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2010

07/15

POSTED BY

petraann

CATEGORY

Environmental Health
Research

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