Hello,

The most powerful, reliable and up-to-dated system is CRISPR/CA9 system.

Check links below:

https://www.addgene.org/CRISPR/guide/

http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html

Many thanks Amin! I would prefer to have a knockdown system rather than the others.. 

Hi,

You can use a lentiviral vector which encodes an shRNA for the target gene leading to its knockdown. We use these lentiviral vectors (pLSLPw) in our lab which also carry a Puromycin resistance gene helping us to select the transfected cells and clone an shRNA under the control of a strong (H1 RNA) promoter. 

Hi,

Do you want to knockdown or knockout? If you would like to knockout a gene, I recommend you to use the CRISPR/Cas9 lentivirus system. It works great; it is really easy and efficient (in cell lines).

If you would like to knockdown, you can use lenti shRNA or siRNA (by transfection or viral infection).

For selection of your transduced or transfected cells I strong recommend you to have a selection marker as GFP or RFP to be able to look at the KO/KD in the specific population by flow (and you can also sort these cells).

If you’re doing CRISPR keep in mind that knockout may take a while.  One of the genes we tested, we observed great KO 7 days post transduction (in cell line).

Good luck!

Hi Ajinkya, thank you!

I was thinking of another Lentiviral vector (pGFP-C-shLenti) contains U6 promoter  - this from OriGene. But wondering if this is an only one step process (direct plasmid transfection), or there will be an essential need to co-transfect this plasmid with other element(s)?

Hi Ana, Many thanks!

Indeed, this lets me think if that the knockout might take this period of time to achieve, that could be an advantage over the stable knockdown itself! The stable knockout take at least two weeks with careful selection and subsequent procedures - this normally extends to 3-4 weeks.

Yes, I'm mainly using human cancer cell lines.

Have you done a knockout before? Any further recommendation would be great!

As I understand, you will need to co-tranfect HEK293T cells with the lentiviral vector and packaging plasmids. After 48 hours collect the supernatent, centrifuge it and filter through a 0.25µm filter. You can infect your target cells with this virus conditioned medium. 

Many thanks Ajinkya! This really helps!

So you think that the supernatant now contains the viruses with target (shRNA) - plasmid material. Then, the virus will helps penetration the genomic materials into the cytoplasm (for the cell of interest), whereas the shRNA materials are prepared for the genomic integration?

We usually use pLKO.1 system and pGIPZ to get stable KD cancer celll lines, and pTRIPZ to achieve inducible KD, I hope them work for you.

I agree with Ajinkya. If you want to elevate the titer of the lentivirus to improve the infection effeicacy,you can further use ultracentrifugation to concentrate the supernatant contaning lentivirus.

It turns up that we need a license to use Lentiviral systems, which we don't have at the moment. But I'll investigate further to have a look at the other systems we could use to produce a stable gene knockdown. Thanks guys! 

Lei Zhou, thank you! interesting systems, and I'll have a look on these.

Zhu and Ajinkya many thanks! Would you be able to provide more details/protocol  about the work you done using the mentioned knockdown procedures? - providing references would really help!

The lentivirus packaing procedure  depends on the lentivirus plasmid and transfection regeant you used. the general procedure is all easily searched on the web.After that,you need to test the titer.about this ,you can read the lentivirus system manual of Life Technology.They will give you enough and professional suggestion.

Thanks Zhu! will check that, and hope everything will go fine..