[Reu_journal] June 13

[Tracy fanous]

Today I met John, Dr. Miskevich's graduate assistant, who introduced me to 
the process of electroporation.  I worked with him for about an hour before 
he left for the day.  The remainder of the day was spent practicing the 
process he taught me.

First, we took capillary tubes and made microscopically fine-tipped glass 
needles by using a "glass puller" machine (which was very cool!).  The 
machine relies on heat and a weight dropping at a specific speed to alter 
the shape of the tube, pulling the capillary tube apart and creating two 
needles with incredibly fine tips.  One needle is then typically filled with 
DNA for injection, but for practice, I simply used a green dye which I 
inserted into the open end using a syringe.  The needle is then held steady 
by inserting it into a contraption that allows it to be manipulated in three 
dimensions.  The very, very tip of the needle has to broken off so that the 
dye can be injected into the specimen.  Believe it or not, this was the most 
difficult part of the procedure due to the extreme delicacy--even lightly 
touching it can break off too much.  I spent a considerable amount of time 
preparing and replacing these needles.

Next, I selected my latest victims--er, specimens.  After dropping them into 
anesthetic to put them into a deep sleep, I placed them in a very shallow 
amount of water in a petri dish with a plastic wire bottom to keep them 
stationary so that I could then inject the dye into the tiny ventricle in 
the midbrain.  The dye is forced out of the needle into the ventricle by 
using a very small puff of air from an air compressor.  This step (the 
introduction of foreign material into the organism) is called transfection.

Once the dye was injected, I then transferred the specimens to another petri 
dish containing a wet kimwipe (so they would not dry out) so that I could 
perform the electroporation using very small platinum wires lightly placed 
on the specimen's head.  Electroporation is essentially the use of a 
low-voltage shock to overcome the normal electrical conductivity of cellular 
plasma membranes, forming small pores in the membrane which allow 
extracellular components (i.e., the dye, DNA, etc.) to enter the cell.

At this point, I finally finished with the rough treatment of the specimens 
and transferred them to a "rehabilitation container" complete with an 
aerator to provide them plenty of oxygen so that they can (hopefully) revive 
and convalesce.  I was happy to see that several of them woke up and were 
swimming around.  Once I am using the DNA, the specimens will be allowed to 
grow for another 24 hours before harvesting them to study the effects of 
different stimuli.

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