I would like to stably knockdown my GOI in the E0771 cell line (mouse breast cancer), which I will then inject into mice to establish a tumour-bearing model. However, I have never done this before and also don't know a lot about the techniques involved. So far, I figured out that I need to transfect my cells with a viral vector expressing either shRNA or miRNA targeting my GOI, and that the viral vector I use needs to be able to integrate into the host genome.
Is this correct? What would be the best option for me to use? What steps/techniques does this procedure require (to generate a vector like capable of this)?
The vector also needs to have some kind of reporter in as well (eg GFP) because I need to be able to visualize tumour growth and metastasis over time (live animal imaging).
Dear Tanja,
Hope this article will help you with your experimental plannings and procedures.
Sigma aldrich now launching interference rna technique. Guidelines given in it will help u to get detail of this projrct.
Where to Buy
There are a lot of companies that offers pre made lentivirus containing shRNA for a target gene.
Sigma (http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/individual-genes.html)
Origene (http://www.origene.com/shrna/),
Santacruz (http://www.scbt.com/research/sirna_and_shrna_plasmid_and_lentivirus.html). (Although I never order anything from Santacruz)
If you want to go the economical way then you can order 2 validated shRNA (if avaliable) from Sigma as bacterial glycerol stocks (~100$/shRNA) and can generate lentivirus in 293 cells using packaging plasmids (http://www.addgene.org/lentiviral/packaging/#3rd )
Selection marker
Generally these viral vectors have a positive selection marker like Puromycin for Sigma (PlKO.1), It depends on where you want to order from. I generally don’t prefer GFP for selection, as a few old reports mentioned that GFP can be toxic. Selection marker like Puro works.
In Vivo Imaging
Fire fly Luciferase is generally preferred for In vivo imaging, You may have to transduce your cells again with a commercial viral vector after you obtain knockdown cells (if you have a luc vector you may generate the virus too). Make sure you have a different antibiotic selection marker for these (like blasticidin, G418, Hygromycin).
Thank you so much everyone for all the advice so far!
@ Deepak Kanojia, you mentioned under in vivo imaging that I might need to transduce my cells again after knockdown, can you please elaborate why? I'm not quite sure I follow. Is it to include the reporter? Also, why is luciferase better? I was under the impression that something like GFP gives a brighter signal which is then better for imaging in whole live animals.
So first transduction is to knockdown the GOI, after you get the knockdown (confirm by western/flow)
Second transduction is to generate Luciferase positive cells for imaging of these cells to assess the metastatic capabilities.
(If you get an shRNA construct with GFP you may not have to do Luc Transduction, provided you have Fluorescent imaging system, http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/trc-shrna-products/shrna-controls.html )
GFP is bright but less sensitive than luciferase, heres the reference (http://www.biotechniques.com/multimedia/archive/00010/03355rr02_10124a.pdf)
While all are viable solutions, I would suggest you look into the CRISPR/Cas9 system. There are a number of papers recently published in which KO cell lines for their GOI's are generated in less than 2 weeks. No lentivirus needed.
Take a look at the Nature Protocols paper linked below. It explains the method, details the process, and the materials needed quite clearly.
http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html
Good luck!
An additional comment: If knocking down your GOI negatively affects cell proliferation, viability, etc.; or you wish to knock the GOI down during development in a spatial and temporal manner; you may wish to use an inducible shRNA expression system. There are different types of systems. One is the tetracycline-inducible shRNA expression system [ref: Nature Protocols 2, 3257 - 3269 (2007)].
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