Hi there,
I would like to induce some genetic diseases (Freidrichs, Duchenne etc.) in vitro, this is however my first time trying such a thing. Are there reasons to choose shRNA, siRNA over Cas9 (or other techniques) and if so, what are they?
I would assume that genetically modifying my cells (my preliminary cell line will be C1C12) is the most straightforward and most consistent approach, I however may be wrong here.
Any insights or helpful tips are welcome!
- Jelle Bonthuis
Dear Yiyang,
1. Yes, the preliminary disorders are Freidrichs Ataxia (reduced expression of frataxin as there are more GAA triplets) and Duchenne (mutated form of dystrophin which has functional deficits). A paper on people affected by Duchenne indicated a significant difference in several other genes as well. However for the purpose of these models I will begin with just the main mutations and possibly mimicking physiological environments.
2. So for Freidrichs we have reduced gene expression and in Duchenne there is a functional deficit which leads through molecular pathways to reduced expression in other genes.
3. As I understood, I will be able to use skeletal muscle cells (specifically from the upper leg) from people affected by Duchenne. We will be working with our university hospital and I am unsure if Freidrich's patients are present.
4. The purpose is, unfortunately not, to investigate new mechanisms or genetics of these diseases. Instead I will be trying to develop in vitro models which should be simililary affected by medication as in vivo cells. In our case specifically the use of iron-scavenging antioxidants.
5. I will ask as I don't know that much about any of this (yet). I am a bachelor student and wanted to do a little more than just the standard internship for my thesis and luckily this project was something the department wanted to do but did not have time for. However, except for my coordinator who will be teaching me how to set up cell cultures and point me in directions, I have been told I will be doing almost everything myself (which is nice but a little overwhelming). That is why I am asking these questions here because I felt this, together with loads of literature and protocols, was the best place to start.
Thanks!
- Jelle Bonthuis
Thanks! I may have not explained myself properly though, the main aim is to create (through induction of the mutation or at least the expression (ShRNA) a replicable method to produce an in vitro model of these diseases. Not to use ex vivo cells so I imagine it would go like this:
Skeletal muscle cells (healthy) > Crispr/talens/ShRNA > skeletal muscle cells (duchenne genome). However the challenge most likely lies in getting these cells to act the same way as Duchenne muscle cells do when in the body (as to accurately test treatments etc.) As such I was wondering if RNA methods or Cas9 differ in their eventual effect (my intuition would say that there isn't a difference, but as I know very little I may be wrong).
I believe addition of dilutions similar of those occurring in the muscle tissue will be needed (Duchenne's dystrophin mutation introduces inwards calcium leaking, which may swell the mitochondria and damage the tissue).
While I have my bachelor thesis in the back of my mind, most of this will be done in my own free time and not part of it, as such I may work on it for longer amounts of time. I am pretty sure I am being overzealous here. But I will try to convince my coordinator (it is his "pet" thought experiment) to let me only work on the Duchenne model as comparing the phenotypes will be easier with patients' samples.
Thank you very much for answering and giving me tips, it is much appreciated!
Happy holidays,
- Jelle
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