Jenise Gordon
Girls Preparatory School
Biology/Forensic Science Instructor

Biomedical Optics Laboratory
Josh Beckham, Ph.D. Candidate

During 4-week research experience I worked directly with Josh Beckman whose research focuses on collateral damage that can occur during cell lasing.  We worked specifically with the NIH 3T3 cell, which is an immortal mouse fibroblast created by the National Institutes of Health.  These epithelial cells were transfected to create two different cell lines.  One cell line was transfected with the pGL3 vector which always has the luciferase gene turned on. The other cell line was referred to as Heat Shock Protein 70 (HSP70luc) because the HSP70 gene was “tagged” with the luciferase gene. Luciferase (from Lucifer meaning “light bearer”) genes produce light by oxidizing the pigment luciferin with the help of ATP. Heat Shock Protein genes are turned on in varying degrees depending on the amount of heat damage experienced by the cells. These proteins unravel denatured proteins and rebuild them in an attempt to save the cell.  We were examining the degree to which these heat shock proteins were produced by the cells.

We used both of these cell lines to determine the amount of heat shock damage caused by lasers. Very little literature exists on laser induced heat shock to cells, so we were working in an unexplored area of science.  The laser of choice was a HoYAG laser which emits a wavelength of 488 nm.  This laser light had to be channeled into an incubator using an optic fiber because we were lasing live cell cultures. The laser energy (mJ) and duration were completely controlled in our research and could be varied to obtain desired results. Cell plates could be easily navigated above the optic fiber inside the incubator using a computer-programmed mechanical stage.

For two weeks we worked specifically on a fun, engaging project using all of the skills we learned in our introduction to the lab.  Our task was to create a smiley face using the HoYAG laser and the two different cell lines.  Attention had to be paid to energy levels and duration for each trial as well as confluency of the cells in the Petri dish or 6-well tissue culture plate.  In order to accomplish this task we had seed new cells each evening, check for confluency, select appropriate plates, operate the HoYAG laser, determine parameters for each experiment, write programs for the mechanical stage, lase the cells and finally image the cells using a special camera.  Immediately before imaging, luciferin was added to each cell plate which reacted with the luciferase gene to produce luminescence. Below are pictures of our best results.

                

           Figure 1. HSP70luc cell line                        Figure 2. pGL3 cell line                              

As seen in Figures 1 and 2 different results were obtained from the different cell lines.  When cells containing the HSP70luc gene were lased, the heat shock resulted in an increased production of HSP70.  Because the HSP70 genes are “tagged” with the luciferase gene, the lased areas were luminescent when imaged.  Inversely, the pGL3 cells always have the luciferase gene turned on, so the areas lased were subjected to large amounts of energy to purposely kill those cells.  As a result, the areas that were lased were the only areas that were not luminescent when imaged.

This is merely a highlight of our 4 weeks experience, since we also had the opportunity to transfect a new cell line with a Luciferase tagged to HSP40 for our PI’s next venture.  We also ran gel electrophoresis, cell titer blue assays, calcein idodine/ethidium bromide viability tests, toured the DNA analysis lab and watched a several cell sorts.

As a result of this research I am developing a biotechnology module where students attempt to use genetic engineering skills to solve a challenge question about Cystic Fibrosis.