I want to clone out a particular ALL cell line: RS4;11 after lentiviral transduction (with no puromycin/similar resistance gene unfortunately) to isolate clones with varying expression of my ectopically expressed gene and to isolate only expressing cells. Previously, with other leukaemia cell lines transduced with various viral constructs, I have cloned them out by limiting dilution (only seems to rarely work), or by using semisolid agar medium. I plan to attempt both of these techniques again, but was just wondering if anyone has any other tips or techniques they might be willing to share? As far as I can see from the literature, there have been no previous attempts to clone out this cell line experimentally.
Important notes:
My viral construct has EGFP, but no puro/similar resistance gene
I normally start cloning cells out approximately 3 days post transduction
Post transduction, you could sort your cells by flow cytometry into cell groups that produce high, low or medium intensity of GFP fluorescence.
To further clarify your issue, is the EGFP driven by an IRES or a different promoter? If it is driven by an IRES module, that is contiguous with your gene of interest, then GFP intensity would likely indicate the expression of the gene of interest. If GFP is driven by a different promoter, I would suggest cloning an IRES-GFP downstream of your gene of interest in the viral vector and then try the clonal isolation.
Given the constraints of your system, I would think that this is the easiest method to isolate your cells.
Hi Sanath,
Thanks for your answer - my vector does indeed have an IRES-GFP. Flow sorting is a strategy I have considered, but I prefer the cloning option in order to have 'purer' populations. Also, time and expertise on our flow sorter is not readily available, so iI would prefer to avoid it.
If you are ok with doing some plasmid cloning, I would suggest you replace the GFP with Gaussia Luciferase or Nano luciferase (both are secreted luciferases). In that case, you can plate the cells, do a clonal isolation and just sample the media from all the wells for the luciferase signal. Depending on the luciferase signal, you could classify your clonal isolates as high, low or medium expressers of the gene of interest.
I would suggest you plate your limiting dilution cells on a stromal cell line such as OP9 or other bone marrow derived feeder lay obtained from ATCC or make you own BM stromal cells. The stromal cells provide a niche for the leukemia cells and increase clone survival. I use this often with good results.
Check out if any of the "ClonaCell" products from STEMCELL can be adopted for your purposes
http://www.stemcell.com/en/Products/All-Products/ClonaCell-FLEX.aspx
http://www.stemcell.com/en/Search.aspx?ts=ClonaCell
Dear all - just an update - the RS4:11 cloned out fine in limiting dilutions in 96-well plates (although they did take 4 weeks!) and also semisolid media made with low melting point agarose. Thanks for your help.
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