4 replies to this topic

[greggers]

Hey everyone,
Ok, so I'm having a little trouble understanding why I'm using the controls I've been using.  Transfection is quite a new technique for me.  The theory behind it is pretty simple, but it's performing it effectively that's got me in a bind.   I feel it's because we're going a little overboard with the controls, and I'm not understanding all of the variables (I could be wrong).

So, what we've done is transfect HeLa cells with the pCR3 vector (Gateway cloning) and our gene of interest (GOI), infect the cells with a recombinant HSV-1-GFP, and then measure viral replication via the fluorescence of the recombinant HSV-1-GFP.
Here's what we have in our plate:

1. No cells transfected
2. Cells transfected with pCR3-GFP
3. Cells transfected with just the pCR3 backbone and no gene
4. Cells transfected with pCR3-ccdb (the destination vector)
5. Cells transfected with pCR3-GOI
6. Cells transfected with pCR3-mutant GOI

Each is performed in triplicate with and without viral infection.  The results are normalized to 4 and 5 above when the background fluorescence (ie. non-infected cells) is subtracted from the infected cells.

What I don't understand is this:  why use the purely the pCR3 backbone AND the destination vector, and why is it convenient to normalize to either of these?  Why not just normalize to the non-transfected cells?  Also, what's the point of transfecting with GFP?  Is it just to show that the transfection worked?

The post-doc I work with is brilliant, but I think struggles to explain things concisely to me

Edited by greggers, 15 April 2013 - 01:43 PM.

[bob1]

greggers, on 15 April 2013 - 01:43 PM, said:

Hey everyone,
So, what we've done is transfect HeLa cells with the pCR3 vector (Gateway cloning) and our gene of interest (GOI), infect the cells with a recombinant HSV-1-GFP, and then measure viral replication via the fluorescence of the recombinant HSV-1-GFP.

Is this correct - you infect with a HSV-GFP and then somehow measure a change in GFP with the new recombinant (i.e. viral replication so more GFP produced)?  I take it from this, the initial HSV-GFP is replication deficient? Or is it that the gene being transfected affects the viral replication somehow, but is independent of the virus?

Quote

Here's what we have in our plate:

1. No cells transfected
2. Cells transfected with pCR3-GFP
3. Cells transfected with just the pCR3 backbone and no gene
4. Cells transfected with pCR3-ccdb (the destination vector)
5. Cells transfected with pCR3-GOI
6. Cells transfected with pCR3-mutant GOI

Each is performed in triplicate with and without viral infection.  The results are normalized to 4 and 5 above when the background fluorescence (ie. non-infected cells) is subtracted from the infected cells.

What I don't understand is this:  why use the purely the pCR3 backbone AND the destination vector, and why is it convenient to normalize to either of these?  Why not just normalize to the non-transfected cells?  Also, what's the point of transfecting with GFP?  Is it just to show that the transfection worked?

The post-doc I work with is brilliant, but I think struggles to explain things concisely to me

I would leave out the destination vector, unless you have this in the GOI transfection as a contaminant somehow.  You need to normalize to a transfection as the transfection of an empty vector by itself could affect the viral replication.  The GFP one looks like it is only to show that the transfection worked, though it could also be used to show that a non-target gene doesn't have the same effect as the GOI, though personally I would do this with a scrambled version of the GOI or something similar.

[greggers]

bob1, on 15 April 2013 - 09:05 PM, said:

greggers, on 15 April 2013 - 01:43 PM, said:

Hey everyone,
So, what we've done is transfect HeLa cells with the pCR3 vector (Gateway cloning) and our gene of interest (GOI), infect the cells with a recombinant HSV-1-GFP, and then measure viral replication via the fluorescence of the recombinant HSV-1-GFP.

Is this correct - you infect with a HSV-GFP and then somehow measure a change in GFP with the new recombinant (i.e. viral replication so more GFP produced)?  I take it from this, the initial HSV-GFP is replication deficient? Or is it that the gene being transfected affects the viral replication somehow, but is independent of the virus?

The HSV-GFP can replicate just fine, and our GOI is independent of the virus.  We've performed genome studies and other screens to understand the relationship between mammalian proteins and viral replication.  Our gene is somehow linked to the replication process -- either a receptor for entry into the cell or the signaling pathway promotes the viral replication itself within the cell.

[bob1]

OK, then your strategy should be fine, I was just trying to get the general gist of the experiment so that I knew which controls were appropriate.

[greggers]

Long overdue thank you -- but thank you!  It seems so obvious now

Edited by greggers, 09 May 2013 - 04:18 AM.