What is the best cell line to use for this experiment?

[SyntonicC]

I am designing an experiment in which I'd like to test the interaction between two proteins implicated in Alzheimer's disease. There's a lot I don't know about cell culture work and experiment design which will probably be apparent so I understand that some of my current ideas may not be the best approaches.

I'd like to do this in one of the transgenic mouse model strains used in Alzheimer's disease studies (typically containing a mutant form of human APP). I would also like to do this study in vivo but I need some kind of immortal neuronal cell line to test the various interactions. The reason is that ultimately my readout will be a misregulated process in Alzheimer's disease (tau phosphorylation, microtubule dynamics etc) and how these processes changes due to the expression levels of the proteins involved. So I need a neural cell because the proteins will not be expressed in other cell types. I need to do this in vivo because there are other player in this pathway and I don't think it's possible to reconstitute 6-7 proteins and their outputs and see how they interact.

Would using primary tissue derived from the hippocampus be a better route? The only thing I am concerned about is not having enough cells for the kind of experiments I will be doing (such as immunoblotting and Co-IPs).

I initially considered PC12 but it appears this is derived from rat so that's not going to work as I have been unable to find any rat Alzheimer's models. I have also read of some mouse neural-derived cell lines such as CATH.a, N1E-115, NB4 1A3, Neuro 2A, and NT4 for example. Would any of these be appropriate?

Thank you for your time!

[relaxin]

You can use SH-SY5Y neuroblastoma cells (available from ATCC). They can be induced to neuronal differentiation with all-trans retinoic acid.

[SyntonicC]

I think I need to give you some more information. In this system, I am interested in observing the interplay between the proteins DAPK and Pin1 and how their interaction affects microtubule dynamics within an Alzheimer's disease model. My original idea was to have various mutant forms of Pin1 expressed where DAPK is either overexpressed or knocked down. To achieve this, I thought it would be possible to cross a transgenic hAPP mouse strain with a DAPK-/- or DAPK overexpressed strain and then transfect in mutants of Pin1 for my experiments. I only want the mutant form of Pin1 expressed so I would have to knockdown endogenous Pin1 expression with siRNA.

I looked up some information on this cell line. As I understand it, these cells would not naturally exhibit Alzheimer's disease characteristics however, I see a paper in which mutant APP was transfected into the cells to induce this phenotype. If I use this cell type, I would have to control the levels and amounts of all three proteins (hAPP, Pin1, and DAPK) through transfection. For DAPK and Pin1, I would also need to regulate endogenous expression levels with siRNA or some other system. I am assuming this would become complicated quickly and may not even work. Given this, is this cell line still the best choice?

Sorry, I am very new to all of this so thanks for your patience!

[relaxin]

SH-SY5Y neuroblastoma cells are the only neuron-like cells I have worked with, and I do not know if they are good for your type of researh. They do not respond to Abeta well, although some papers have been published in this area of research. Perhaps you can do some serach in PubMed, and find what cells other researchers are using.

[SyntonicC]

Thanks, for your help. I have been doing a literature search but I haven't come across any papers so far that have done anything quite like what I am attempting (which just makes me think my approach is not the best one). I will keep looking.

Would it be possible to use primary cells derived directly from hippocampal or cortical tissue? From what I understand, this sort of procedure would not generate nearly enough cells for the kind of biochemical work I am attempting to perform and is not a viable option.

[relaxin]

Primary neural cells are difficult to work with and time-consuming. You may be better to use cell line that can be induced to become neuronal cells. Perhaps you can get some ideas from the following publication:

http://www.ncbi.nlm.nih.gov/pubmed?term=Pin1%20sh-sy5y

[amanc]

Have you thought about using IMR-32? There is a publication looking at IMR-32 as a cell culture model for Alzheimer's disease, see: http://www.ncbi.nlm.nih.gov/pubmed/7884825. It can be a bit tricky to work with though, it is loosely adherent and so will easily detach from plates while handling. But that is manageable with a bit of care.

Most people seem to work with more constructed models, transfecting in key components using lentiviral vectors. For a review of that approach see: http://www.ncbi.nlm.nih.gov/pubmed/15974924.

Good luck!