Protocol Online logo
Top : New Forum Archives (2009-): : siRNA, microRNA and RNAi

How to do stable knock-down by shRNA advices for beginner - (Apr/14/2010 )

Dear all,

I would have some questions regarding a stable transfection with small hairpin RNA (shRNA). We would like to generate of stable (long-term) knock-down of target gene expression in a cell line. There are probably many systems for stable tranfection from different companies. We have different siRNAs (used for transient knock-down) that are strongly inhibiting expression of target genes. We want to use the sequence from these siRNAs for designing shRNAs. The shRNA, we would insert in vector (e.g. pSilencer 4.1-CMV neo) and express in cells treated with antibiotics. We want to use plasmid based shRNA strategy, instead of viral vectors delivery.
I would like to ask you, do you have any experience with such a kind of experiment?
Could someone give me a useful advices how to do stable knock-down? For example: Which vector did you use (from which company)? Is viral based vector better than normal plasmid (containing just selection marker)? Did you use siRNA for designing your shRNA, that was consequently inserted in vector and this system was successfully working? Is non-viral vector (plasmid) after transfection into cells also integrated in genome? Could you recommend/give me any detailed working protocol?
Do you have any other suggestions (what should I know, consider before starting with experiment)?

Many thanks,
Vic

-victor.m-

1. The siRNA to shRNA converting (successful) rate may not be 100%, you might want to use some designers to double check.

2. For non-viral vector, I have used pSUPER (OligoEngine), BLOCK-iT U6 and H1TO (Invitrogen), and pRNAi (Biosettia). Invitrogen and Biosettia RNAi vectors are linerized and OligoEngine's pSUPER is sold as circular plasmid. The linearized vectors give you much lower background (i.e. inoculate 2-3 colonies and you might get them all right). Personally I favor Biosettia's vector since only one DNA oligo is required to make shRNA clone, it's amazing and really high efficiency. I don't use next-door's free RNAi vectors any more because the oligo money I saved from using Biosettia's vector just like I get it for free. So, I don't bother to digest and gel-purify other RNAi vectors since then.

Below is the links for protocols:
OligoEngine
http://www.oligoengine.com/products/psuper...ER_protocol.pdf
Invitrogen
http://tools.invitrogen.com/content/sfs/ma...yvector_man.pdf
Biosettia
http://biosettia.com/download/protocols/bi...rnai-manual.pdf

3. The shRNA viral vector especially lentiviral shRNA vector overcomes the transfection issue since quite a few cells are hardly transfected. But the lentiviral packaging is a key, you better good on packaging. A lot of people use pLKO.1 since it's free but I am quite loyal to one oligo shRNA method to save time.

4. Linerize your non-viral shRNA vector before transfection, the non-homolog end joining and homologous recombination should give you multiple copy integration. check the protocols listed above.

Good Luck.

-Functional Screens-