Cell Culture days 3 & 4

Yesterday, I again changed the media on my cells and then added in the mixture that, hopefully will allow me to measure the response of the estrogen response element (DNA) to different amounts of estrogen.   Picture this: my cells are growing on the bottom surface of a little well.  Layered over the cells is a pool of pink medium that will provide the nutrients that the cells need to continue to grow.  I prepared a mixture of 4 substances that when added to the cell cultures will cause specific pieces of DNA to enter the cells (scientists say transfect).  First I put a small amount of medium (it looks like water) into a tube.  Then I added a small amount of liquid that contained pieces of DNA with the estrogen response element (ERE) next to the gene for an enzyme called luciferase from fireflies. Next, I added another liquid that contained other pieces of DNA that with the gene for luciferase from a sea pansy (that’s a soft coral for those who know invertebrate animals).  Finally, I added lipofectamine.  The idea is that the lipofectamine (which is molecularly similar to the lipid molecules in the cell membrane) will help the bits of DNA (called plasmids) across the cell membrane of my cells.  I have practiced genetic engineering!  I returned my culture plate to the 37⁰ C (98⁰ F) incubator.  The cells, plasmids & lipofectamine “cooked” together overnight and we assume tranfection has occurred. 

==== ERE ERE ERE firefly LUCIFERASE GENE ====== (really in a circle) 

==== sea pansy luciferase gene === (also in a circle) 

Today, I again changed the media on my cells and this time I added different concentrations of estradiol to 6 sets of 3 wells each (0, 0.01, 0.05, 0.1, 0.5, and 1.0 nM) and then returned the dish to the incubator.  Tomorrow will be the moment of truth.  The way this technique works is that:  estrogen binds to the ERE and turns on the nearby luciferase gene.   Then the cell makes mRNA and then luciferase protein.  Later we’ll provide a substance (scientists say substrate) that luciferase will convert into another substance that makes light.  And we’ll measure the amount of light which will tell us how much luciferase was made.  My hypothesis is that the lower concentrations of estrogen will correspond to lower amounts of light.  By the way, remember this is from a firefly (or lightening bug).  The chemical reaction is how fireflies make light!   The reason that the sea pansy (Renilla) luciferase plasmid is added is as a control.  Tomorrow we’ll use a machine that adds the substrate and measures the amount of light.  The machine will first measure firefly-luciferase-produced light and then adds another liquid (Stop @ Glo) that stops the firefly reaction and adds the substrate for the sea pansy luciferase reaction.  Then, the machine will measure the light from the second reaction.  We will have a measure of light from both luciferases in each well and the machine will report a ratio.  This provides an internal control so we can have more confidence in our numbers.   

It is very different being on the student side of the bench; worrying that I won’t do everything perfectly and so contaminate my cultures or otherwise stop the experiment prematurely.    My supervisor is very patient and very clear and allows me to run through what I am to do mentally before I have to do it with my hands.  She’s a very good teacher.  She noticed some things that I have trouble with, like remembering the next step or how much to put in, so she taped the steps on the outside of the hood so I can see them easily from where I’m working.  And, we’ve modified the standard procedure (biologists say protocol) to a way that is easier for me to accomplish without mistakes.   My hands shake with a long pipette but with shorter ones I’m doing fine.  It’s a longer process but has contributed to my success.  I can emphasize more than ever with my students when I ask them to do something unfamiliar.   My cells show no sign of contamination!  I seem to be getting better at working with very small quantities and with great care to avoid contamination.  I enjoy the feeling of a challenge met.  We’ll see if I can get the expected curve from the machine tomorrow.