5 replies to this topic

[Katie Z]

Hi Everyone,

Recently, a friend of mine approached me for some technical help in tagging her osteosarcoma cells with GFP. One of her friends recommended her to use the Retro-X Universal Packaging System and pRetroQ-AcGFP-N1 cloning vector from Clontech for her. When I talked to her to understand her purpose her project and downstream applications, I felt she don't really need such an expensive system to do a simple GFP-tagging.

Her background is in tissue engineering and possess no idea how such work can be done. She just need her cell lines to be tagged with GFP so that she'd be able to view the cells under a florescence microscope. I'm thinking she might just need to transfect her cells with normal GFP-tagged vector, as I supposed she's doing this as a positive control. However, I realised that she actually need to inject these GFP-tagged cells into mice to track the movement of the cells and she's not doing any sort of gene expression work. As I ask around, it seems that she needs to create a stable cell line with GFP prior in injecting the cells into an animal system.

Now the question is, how can I go about in doing this? It seems to me that it's just more than just transfecting the GFP-vector into her cells. As I search around, most GFP vectors are cloning vectors and are quite expensive for such experiment (I think). I wonder is there some other vector/plasmids tagged with GFP for such work, or some other methods that's feasible.

Need advise for this.

Thank you very much

Edited by Katie Z, 22 September 2010 - 06:10 PM.

[bob1]

So long as the plasmid is a mammalian expression plasmid, and has a selectable marker such as hygromycin or zeocin that will kill eukaryotic cells, then it is possible to create a stably expressing cell line.  

With regards to expense - she could subclone the GFP into an appropriate vector, or just find someone with a useful vector and then get them to send her some plasmid on filter paper.

[Katie Z]

Hi Bob1,

Thanks!!!

Well, in actual fact I'm not very good in cloning as I only did it once so far. Not to say about this friend of mine who has zero background. Now I'm trying to ask around and try to assist her as much as possible as it seems to me that her supervisor is not really guiding her well.

As her downstream applications involves animal work, seriously I wonder will transducing a retrovirus with GFP a better option??

bob1, on 23 September 2010 - 01:52 PM, said:

So long as the plasmid is a mammalian expression plasmid, and has a selectable marker such as hygromycin or zeocin that will kill eukaryotic cells, then it is possible to create a stably expressing cell line.  

With regards to expense - she could subclone the GFP into an appropriate vector, or just find someone with a useful vector and then get them to send her some plasmid on filter paper.

[Rsm]

You can also use a cell tracker dye for this, for example CFSE or DiI. That stuff links covalently into your cells and labels them for at least two weeks or such (depends on proliferation). It's fairly simple, and not so expensive (for ~$100 you can make 200 labelings). Haven't tried other dyes, but can recommend particularly DiI.

Cheers,

rsm

[Katie Z]

Rsm, on 23 September 2010 - 11:52 PM, said:

You can also use a cell tracker dye for this, for example CFSE or DiI. That stuff links covalently into your cells and labels them for at least two weeks or such (depends on proliferation). It's fairly simple, and not so expensive (for ~$100 you can make 200 labelings). Haven't tried other dyes, but can recommend particularly DiI.

Cheers,

rsm

Thanks Rsm, but I wonder will this method feasible when the cells are to be injected into an animal model?

[Rsm]

Sure, that's what they are made for... Here, "Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking" PMID:18396458, six weeks after injection. Lots of those papers...

cheers,

rsm

Edited by Rsm, 24 September 2010 - 04:22 AM.