It’s hard to believe that I’m already in my third week in
the lab! Every day is always so exciting and eventful that the days go by so
quickly and before I know it, I’m almost halfway done!
My second week in the lab was even better than the first.
Monday, I received my samples from the NAPCORE (the sequencing group at CHOP)
from the two different PCR purification kits that I ran the previous week. I started with 4 wildtype zebrafish embryos for
the MLL gene, extracted their DNA and then ran PCR with each of the four
samples. The four samples were then
divided into two groups- a gel-purified group and a PCR purified group. In the gel-purified group, all 25ul of the
PCR product was run on the gel and then the band was cut and the DNA was
sequenced from the gel. In the PCR purified group, only 5ul of the PCR product
was run on a gel, just to make sure that the PCR worked, and the rest of the
PCR product DNA was used to purify from the tube. Because the PCR purified takes a lot less
time (you don’t have to cut every band from the gel), we were hoping that it
would have the same quality sequence as the gel purified. Unfortunately, the
PCR purified sequence was a mess and not readable, so we have to stick to the
gel purification method and cut all the bands from the gels. I also helped
Patrizia genotype her embryos, as wild type, heterozygous, or mutant, from her
PCR sequences. We did this by looking at
the PCR sequence and finding the region of the MLL gene that was of interest to
us. Once we found the region, we looked to see if the chromatogram at a peak
for a G (wild type), a peak for an A (mutant), or two peaks for an A and G
(het). Once we had the genotypes of each
embryo, we went back and looked at the pictures that Patrizia had taken of the
embryos and tried to draw conclusions based upon the number of cells that were stained for
(meaning they were expressing) a specific transcription factor and their
genotype.
Tuesday was a great day, not only because I started one of
my bigger experiments, but also because Dr. Peretz came to visit! In the
morning, I helped Patrizia finish genotyping and draw conclusions about the
remaining embryos that we started yesterday, and then we spent the remainder of
the morning going over everything behind the experiment that I would be
starting later that day. My project for
the next week or so is to perform a whole-mount in situ hybridization (WISH)
experiment for embryos that are 8-10 somites, 18 hour post fertilization (hpf),
and 24 hpf. I started with 25 embryos per stage to go through the WISH
procedure with. In this procedure,
through many washes and several incubations, the embryos are stained with a bluish
purple color in the cells that are expressing a specific, or in my case the
specific transcription factor, lmo2.
Lmo2 is expressed in hematopoietic progenitors, erythrocytes, and endothelial
cells. Lmo2 has also been showed to be
an embryonic lethal, meaning that targeted knockout the whole gene leads to the
blood cells not developing properly in the embryo and eventually death. There
are several hypotheses about the complete role of lmo2. First, it is possible that lmo2 is required
for maintaining the hemangioblast population and for the differentiation of the
hemangioblast into different types of blood cells. The second option is that
lmo2 may work with other transcription factors that also promote differentiation
and all three are required for complete development and full differentiation of
the blood cells. The knockout of lmo2
also affects both primitive and definitive hematopoiesis. Although lmo2 expression occurs in many
different parts of the embryos depending on the stage of development after
fertilization that the embryo is in, for my experiment, I am mainly interested
in the posterior (tail region) of the embryo and how many cells in that region
are expressing this transcription factor.
The long-term goal of this procedure is to detect an mRNA of
interest. To do this, in the WISH
procedure, I will be using an anti-sense RNA probe, which is a complementary
strand to the RNA that is of interest. Through the annealing, the anti-sense
probe random flags some of the Us in the RNA with a DIG protein group, which is
used as a marker. Then, we use an
antibody, which is an anti-DIG molecule, then attaches to the DIG markers on
the mRNA to mark where the DIG groups are. Following the marking, a salt and a
part of the antibody interact to form a blue substance, which is the blue
staining. Because the embryos are
gradually moved from different substance environments, the procedure takes 3
days (which in my case was Tuesday, Wednesday, and Thursday). Although it was
fun to wash the embryos in PBT and methanol, the fun day was Thursday. After the embryos were in the staining
solution, I left them on the rocker so that the staining (the reaction of the
salt and the part of the antibody to create the blue color) would occur more
quickly. We periodically checked on them
over the course of 2 hrs until they were stained just enough so that the cells
expressing the mRNA were visible, clear, and able to be counted. I then stored
them in 4% paraformaldehyde over the weekend so that I could start imaging the
embryos when I came in on Monday.
My lmo2 WISH embryos on the rocker during the staining
Back tracking a little bit to Tuesday when I got my assignment, after Patrizia explained the experiment to me, I headed off
to Shake Shack to meet the other EXP kids in Philly. It was great to see
everyone and hear about all the cool research they are dong in their labs and
even how it is getting use to living on their own! When I returned from lunch,
since the WISH procedure was a real experiment that I couldn’t screw up,
Patrizia showed me all the techniques that I needed for the procedure so I had
time to practice them before Dr. Peretz came.
Dr. Peretz’s visit worked out perfectly because my PI was in the lab
that day so Dr. Peretz got to meet her and the rest of the lab team! It was so nice of her to stop by and check on
me and everyone in the lab loved meeting her! Thank you for coming Dr. Peretz!!
For the rest of the week, I continued with my lmo2 WISH
procedure. The purpose of my experiment
was to look at the staining of lmo2 in the specific mll mutant that we have and
to see if the staining and therefore the expression of lmo2 was different in
the mutant then in the wild type cells. I
thought that this project was the perfect balance for me because the result was
something that no one had looked at before and we weren’t sure what was going
to happen, but at the same time, it was structured enough I knew exactly what
to do and just needed to follow the protocol and I always had Patrizia there to
help if I needed it. At the conclusion
of the WISH on Thursday I was so happy because Patrizia said that my staining
looked great and that I hadn’t lost any of the embryos during the in situ! I
can’t wait until I see the results of my in situ through imaging this week!
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