Bridgid, Entry #3, Something Fishy is Going On Around Here

Last week was my third week in the lab. Just like any other week, it was nothing short of exciting and eventful.  I made big strides on my project this week, and in addition I unexpectedly helped out Abby, the other college student in the lab, and Patrizia with her project when they ran into some trouble in the middle of the week. Let me explain.

On Monday, since Patrizia was in the fish room injecting, Abby and I spent the majority of the morning reading articles that had to do with the expression of lmo2 at different stages of development in the zebrafish and also how to sort cells, which is what Abby’s main focus for the week was.  In the afternoon, I got to have first hand experience in what Patrizia says is one of the most dreadful tasks in working with the fish—imaging.  Imaging is done after the in situ is finished and the embryos were kept in PFA to freeze them in the desirable stage of development.  For my experiment, I started out by imaging the mutant lmo2 18hpf embryos that I performed the in situ on.  Because the 18hpf is in the middle of the other two stages of development that I have, (8-10 somites and 24hpf) Patrizia wanted me to start with these to see if I noticed any difference in the staining and then if needed I could image before and after this time point. After starting with 25 embryos for the in situ, I was able to recover 19 that I was able to image.  I took one picture of each embryo, making sure to focus on the posterior end staining because that was the area of interest in which the gene lmo2 is normally expressed at 18hpf.  Although it may not seem like a lot, between getting all 19 good embryos in position on a mini gel under the microscope and then using the computer and the microscope to find the right exposure and focus for each embryo, it took the remainder of the day on Monday and the beginning part of the day Tuesday to image all 19 embryos.  It got a little frustrating towards the end when it was hard to find the right focus for the picture, but it was so cool to notice that between just these 19, we were already seeing differences in the amount of staining or gene expression in embryos. Although we were seeing differences, they didn’t mean anything yet because I was doing the imaging blind, which means that I didn’t know the genotypes of each embryo so that I am not biased to how I interrupt or take the images based upon the result that we would like to see.  After a long day of switching between staring in a microscope and at a computer screen, it was so nice to grab dinner in the city!

Tuesday morning I spent almost the whole morning finishing my images for the 18 hpf lmo2 embryos.  After I was finished imaging, I started the DNA extraction for the first 8 emryos that I imaged.  This was the first time that I started the DNA extraction from single embryos, which was pretty cool! I felt like I was really doing it all by myself, which is a great experience and test in responsibility and concentration.  That being said, because it was my first time doing the DNA extraction starting from the single embryos, Patrizia and I both thought that it would be a good idea to start with just the 8 samples.  Once I put my samples in the water bath to sit over night, I prepared and ran the PCR for the mutant rescue experiment that Abby and I have been switching off working on.

Wednesday was quite the day in the lab! I started in the morning by finishing up the DNA extraction for my 8 embryos and then once the DNA was extracted, I quantified the DNA on the Nanovue to determine the concentration and purity of the DNA.  I then used that DNA to prepare and run PCR on my samples.  Even though DNA extraction and PCR is pretty cool (especially for me), the real agenda for the day was to help Abby with her project.  Today was the big day where we planned on sorting the lmo2 cell line with and without the mutated gene, MLL-GAS7.  Abby and Patrizia spent the whole morning sorting the embryos based upon their type and color of their fluorescence in order to make sure that we sorted the cells correctly.  After walking through the maze of building between Penn and CHOP, we finally arrived at the sorter. The cell sorter this is huge machine that sorts cells based on certain parameters that you set- for example, what types of cells you would like to keep to analyze, how many cells (or events) per tube, and the percentage of cells expressing a the desirable trait that you are looking for, which in our case was a certain type of fluorescence.  Because we had never sorted this cell line before, no one had any idea what to expect in terms of the number of cells we would be getting.  Everything seemed to start out well, after all the controls were in place, things were going as planned, until we started to run the actually samples. The first sample, our control with only one variable, sorted fine, ending with a total of 5 tubes with 9,000 cells in each.  The real problem started when we went to run the mutant sample.  Even though we started out with more embryos in the sample, we ended up only filling 3 tubes with 9,000 cells each and had a lower percentage of expression of the gene that we were looking for, the exact opposite of what we expected.  Because we didn’t know what to expect, we just went with it and Abby and Patrizia went the cytosine in the Main Hospital, where the machine is supposed to produce slides of cells from the culture that you started with so that you can look at them under the microscope.  Except that didn’t happen.  When they came back to the lab, we noticed that not one slide at a single cell on it.  There was absolutely nothing on every slide. 

We were stumped, and spent the remainder of the afternoon trying to figure out what went wrong and why did we not have any cells at all.  Ti was a frustrating afternoon, just because so much of the experiment was machine based that we had no idea where to start troubleshooting. We all tried to think of something, but by the end of the day, we only had one hypothesis—maybe there was something wrong with the sorter, but none of us knew where on earth to start looking for that answer.

Thursday morning I ran by PCR for the first 8 lmo2 samples on a gel, cut the bands from the gel, and then prepared the sequencing reaction.  By the time the afternoon rolled around, although we didn’t have an answer to what went wrong with the cells yesterday, we did have an alternative way look at the cells.  One way that we could try and see the blood cells was to take a fish that was about 2 days old and cut its major blood vessel. Because the fish was already 2 days old, the blood cells were already in circulation, so we would cut the fish and then let it bleed onto our slide.  After the cells were on the slide, we removed the dead fish and then stained the slides in various purple dyes to highlight the cells that would hopefully be on the slide.  Since we weren’t completely sure that this technique would be entirely successful, we only tried it on wild type fish so that we knew what the cells were supposed to look like.  After spending the rest of the day on Thursday and a large part of the day on Friday attempting to perform to the staining in order to have cells to look at, we ended with about 3 or 4 good slides were the cells were visible.  Although we ended with some slides, this method was extremely time consuming and the cells still weren’t the best to analyze. Luckily, by Friday morning, Patrizia had figured out what went wrong with the experiment on Wednesday.  She found out that the speed in which the cell sorter was sorting the cells was too fast for the types of cells that we had so the cells were most likely destroyed in the process of the sorting.  Since we now believed that this was the reason for the failure, we booked a time on the Influx, a gentler machine, for next Tuesday to determine if we would have cells at the end of that sorting process.  Friday afternoon concluded with a few more staining and then a lab meeting, at which both Abby and I presented on the work that we had done in the past two weeks.