The past month has really flown by quickly; it's hard to believe that my time in Dr. Mourkioti's lab is already halfway over. I moved into The Radian two days ago (I needed to sublet a new apartment this month because I had previously mixed up my living arrangement), and it's really nice. They even have free drinks in the lobby. The act of moving into a new apartment for the 2nd half of my internship really makes it feel like a new beginning. It helps me come into
In lab, we've confirmed that NF-kB signaling, which is the pathway I have been studying, gives rise to telomere shortening in the injured control and IKK2ca (in which NF-kB activation is inducible by injury) mice. My lab had previously shown this result, but this time we have more detailed graphs and data. Now that we know there is telomere shortening, as caused by NF-kB signaling, we have to figure out the mechanism of telomere shortening. There are four main possibilities: DNA damage at the telomeres, prolonged muscle stem cell (MuSC) proliferation, epigenetic causes, and RNA damage at the shelterins(these protein complexes "cap" and protect telomeres; they're like telomeres for the telomeres!). We're using different techniques with different markers for each of the aforementioned possibilities: a TIF (Telomere dysfunction Induced Foci) assay makes use of a DNA damage marker called 53BP1, tissue culture techniques are used to quantify MuSC proliferation and to stain MuSCs for the chromatin-modulating protein ATF7, and Q-PCR (similar to PCR) is used to quantify RNA damage by allowing us to analyze the RNA transcripts. We're working on the TIF assay and the tissue culture currently, and we'll do Q-PCR sometime in the near future. Maybe not this week though; our lab gets a break on July 4th and we're also going to eat dim sum on Friday to celebrate my to-be grad student David, who is "graduating" from the lab at the end of the month.
I'm doing more PCR this week as well to confirm some genotyping for the mice. David will walk us through PCR in detail so we become experts on it. Also, last Friday, I got to watch David and the two undergraduate students working on our neurology project. Specifically, after euthanizing the mice, they dissected them and cut out their gastrocnemius (calf) and tibialis anterior (shin) muscles. I was kind of scared to do it since the tissue removal requires great concentration and surgical precision, but I'll have a chance to do it next time. We fixed them in gel using liquid nitrogen and dry ice. This method, called tissue histology, has very strict protocols because one has to freeze the muscles in a way that the cells won't be damaged when placed in a -80 degree Celsius fridge. After we were done, the undergraduate students dumped the liquid nitrogen into a sink and started playing with the "smoke", which was quite amusing to watch.
This week, I'm going to practice tissue sectioning, which uses a machine called a cryostat. Essentially, one uses the machine to cut thin slices of the fixed muscle tissue and imprints them on microscope slides. That way, we can analyze them later.
In lab, we've confirmed that NF-kB signaling, which is the pathway I have been studying, gives rise to telomere shortening in the injured control and IKK2ca (in which NF-kB activation is inducible by injury) mice. My lab had previously shown this result, but this time we have more detailed graphs and data. Now that we know there is telomere shortening, as caused by NF-kB signaling, we have to figure out the mechanism of telomere shortening. There are four main possibilities: DNA damage at the telomeres, prolonged muscle stem cell (MuSC) proliferation, epigenetic causes, and RNA damage at the shelterins(these protein complexes "cap" and protect telomeres; they're like telomeres for the telomeres!). We're using different techniques with different markers for each of the aforementioned possibilities: a TIF (Telomere dysfunction Induced Foci) assay makes use of a DNA damage marker called 53BP1, tissue culture techniques are used to quantify MuSC proliferation and to stain MuSCs for the chromatin-modulating protein ATF7, and Q-PCR (similar to PCR) is used to quantify RNA damage by allowing us to analyze the RNA transcripts. We're working on the TIF assay and the tissue culture currently, and we'll do Q-PCR sometime in the near future. Maybe not this week though; our lab gets a break on July 4th and we're also going to eat dim sum on Friday to celebrate my to-be grad student David, who is "graduating" from the lab at the end of the month.
I'm doing more PCR this week as well to confirm some genotyping for the mice. David will walk us through PCR in detail so we become experts on it. Also, last Friday, I got to watch David and the two undergraduate students working on our neurology project. Specifically, after euthanizing the mice, they dissected them and cut out their gastrocnemius (calf) and tibialis anterior (shin) muscles. I was kind of scared to do it since the tissue removal requires great concentration and surgical precision, but I'll have a chance to do it next time. We fixed them in gel using liquid nitrogen and dry ice. This method, called tissue histology, has very strict protocols because one has to freeze the muscles in a way that the cells won't be damaged when placed in a -80 degree Celsius fridge. After we were done, the undergraduate students dumped the liquid nitrogen into a sink and started playing with the "smoke", which was quite amusing to watch.
This week, I'm going to practice tissue sectioning, which uses a machine called a cryostat. Essentially, one uses the machine to cut thin slices of the fixed muscle tissue and imprints them on microscope slides. That way, we can analyze them later.
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| The cryostat machine used to section various types of muscle tissue, anything from large gastrocnemius muscle to tiny Achilles tendons. The inside of the machine has a temperature of between -16 to -24 degrees Celsius. |
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