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Double transfection questions


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#1 cell_man

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Posted 01 December 2009 - 11:17 PM

Hi, I am trying to transfect microRNA expression clone and target expression clone in to 293T cells. I am planning to transfect the miRNA clone (which contains GFP marker) first and sort for GFP positive cells and after one day, transfect these GFP positive cells with the target vector which contains luciferase marker and hence i will measure luciferase expression. Does this strategy work? or should I co-transfect both plasmids at the same time for the best results i.e. target validation.

Another question is that after sorting for gfp positive cells, can I freeze the cells in Ligquid n2 until I transfect them with target vector?

Finally, how do we select with neomycin in case of transient transfection? because the episomal plasmids wont be passed on to the daughter cells, aren't they all going to die after adding antibiotics?

Please reply

thanks in advance.

#2 madrius1

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Posted 02 December 2009 - 01:00 PM

Hi, I am trying to transfect microRNA expression clone and target expression clone in to 293T cells. I am planning to transfect the miRNA clone (which contains GFP marker) first and sort for GFP positive cells and after one day, transfect these GFP positive cells with the target vector which contains luciferase marker and hence i will measure luciferase expression. Does this strategy work? or should I co-transfect both plasmids at the same time for the best results i.e. target validation.


There are several ways of doing what you want to do. But something is lacking, in my point of view, in what you are proposing here.

First, sorting the cells for GFP expression is good. This way, you'll be sure to work with positively transfected cells and that all the cells do contain you miRNA. This will unfortunately not be true with your second transfection. So, your results for luciferase expression will be biaised by the transfection efficiency. You have to be able to either positively select transfected cells or to correct your results for the transfection efficiency. Here are two ways of doing this.

- First, you could choose a vector that contains a selection marker (neomycin, puromycin, gentamycin, etc) and select the cells.

- Second, you could add a third plasmid that contains any given protein you can easily detect and quantify (B-Galactosidase) to your transfection mix. And when you analyse the luciferase expression, you have to divide each result with the corresponding expression level of B-Galactosidase to correct your result with the transfection efficiency. This way, your results won't be biased by the transfection efficiency.

Another question is that after sorting for gfp positive cells, can I freeze the cells in Ligquid n2 until I transfect them with target vector?


Since your GFP transfection as I understand it will be transient, freezing/thawing/expansion of these cells is not the best way to go. Cells could (and will) eventually loose GFP and your miRNA. I suggest you work will freshly transfected and sorted cells. Or you make up a stable cell line expressing your miRNA/GFP consctruct.

Finally, how do we select with neomycin in case of transient transfection? because the episomal plasmids wont be passed on to the daughter cells, aren't they all going to die after adding antibiotics?


In transient transfection, you do not have to select cells with antibiotics. When you do a stable cell line, you rely on rare events of genome incorporation. It is the only way that the construct will be passed on to the daughter cells.

Hope this helps!

#3 96well

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Posted 02 December 2009 - 03:44 PM

Im my opinion the best will be to:
1) Made a stable transfection with the luciferase vector.
2) Select the clone luciferase-positive
3) Transfect these cells with miRNA-GFP
4) Make FACS sorting (and keeping both GFP negative and positive)
5) Treat both GFP positive and negative cells
6) Measure the extent of miRNA is modulating luciferase activity.

best

96well
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