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SH-SY5Y stable cell transfection and RA differentiation


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#1 Lfs

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Posted 28 December 2013 - 03:21 PM

Hello! 

 

It is my first time working with SH-SY5Y, and I have some questions. I will appreciate any help! 

 

First, I would like to know if low serum (1 - 5%) is able to stop or decrease cell division and, in a transfected cell line, if the DNA can be stable for at least 7 days in this condition (DMEM/F12;  1-5% serum). If it is not possible, for how long the DNA stays stable with high protein expression in a SH-SY5Y line (double time - 48h)? 

 

I ask this because I was wondering if there is another way to make a stable transfection (I just need 7 days of high protein expression) without spending lots of months working on it. I have only 4 months to finish my research wacko.png  and I need to know if there is an easy way to do it. 

 

Second, if I differentiate SH cells with RA 10uM, will they continue to grow or this kind of differentiation stops cell cicle (should I maintain RA until the end of the experiment)?. Can I easily transfect these cells as a second option? 

 

And finally, if there is no way to get rid of the standard protocol, can I use 20% of fetal serum in order to speed up cell growth and get my stable line faster (once sh double population time is 48h)? Right now, I'm using 10% with 600 ug/ul of G418 in DMEM/F12 and I already got some cell colonies (20 days pos transfection in 6 well plates) and now I will try to pick up monoclonal clones.

 

Thanks in advance!



#2 bob1

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Posted 06 January 2014 - 12:16 PM

The transfection is not dependent on serum concentration - though if you are using lipid based methods like fugene or lipofectamine, then you should form the DNA/transfection reagent mix under serum-free conditions.

 

Plasmids are often stable for many days after transfection, but it depends on the cell line and the plasmid being expressed.  You could test this quite easily.

 

The quickest way to make a stable cell line, if you don't really care about the properties of the cells (as you are using SH-SY5Y I'm guessing you do though) is to transfect, select, and then pool all the cells you get out (called polyclonal stable cell lines).  It will still take about a month to do this if you include all the preparation steps like determining the effective selection dose.   As you are probably concerned with the properties of the cells - you should determine the growth rate hasn't changed, look at markers for particular gene expressions, so as to determine that the stable cell line is the same as the parent, just with the addition of the insert (there are number of companies that offer cell line determinant services, but the cost is quite high.  Many journals will require this before publishing too!)






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