Dear Francesco,

Lentivirus is really an efficient way to generate stable cell lines with any other gene ORF expression, shRNA/miRNA and CRISPR/Cas9. Integration into host cell genome, it mediates long-term and stable expression of exogenous genes with a simple system for vector manipulation and production. With broad range of host, lentivirus guarantees efficient transfection in both dividing and non-dividing cells.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

If you have shRNA sequences validated, i think that it's more easier and less expensive to stably transfect all your cell lines, instead of transfect transiently your cell lines before each experiment. Stably transfection generates reproducible results except if the silencing of one gene leads the death of your cells.

To answer to your question in 2), siRNA could be coupled to a marked nucleotide to check transfection efficiency

To answer to your question in 3), The Virus method is easy to learn and to use but you need a P2 laboratory, specific vectors and one packaging cell lines.

Use lentivirus to create a stable cell line expressing your shRNA.

The most important factor to consider when planning silencing experiments is to have a reliable negative control, which has to be a "non targeting" siRNA of choice. According to the phenotype you are going to measure, you might want to avoid to use transfection reagents that you would have to use when transfection shRNA encoding plasmids or siRNA duplexes, and thus you would need to go for Lentiviral-mediated silencing. On the other hand, transduction with Lentiviruses also induces some changes to the cell, which might affect your readout. So my suggestion would be first to compare the two procedures using appropriate negative controls, to see which method affects less your readout.

Coming to 2) the issue of tracking the "transfected" cells, as also NIcolas mentioned you can use labelled oligoes. But you should also remember that if you use Lentiviruses you can also similarly to shRNA plasmids also express GFP/RFP as a marker of transduction. Actually using Lentiviruses is a really easy way to produce stably transduced cells where your gene of interest is consistently silenced. This can be achieved by using Lentiviruses carrying instead of or in addition to the GFP/RFP maker also an Antibiotic resistance marker (for example conferring resistance to G418, blasticidin, puromycin, etc...)

3) Before deciding to start with stable, you should consider if you want to go for clonal or mixed populations, considering that each method will have advantages and disadvantages

4) Working with Lentivurses is extremely easy, but you need a BL2 laboratory, even if only for the production phase of the Lentiviruses, and the tranduction of your cells. Then stable transfected cells can be used in a BL1 environment.

Please keep in mind that it will be advisable to check different sequences targeting your gene of interest and, hopefully by testing 4 different oligoes/plasmids/Lentiviruses you will get at least one with satisfactory levels of knock-down.

Furthermore, if you design your oligoes to target the 5' non coding region of the transcript, you will easily able to check for off-target effects by transfecting (or transducing) the cells with the gene of interest (or mutants thereof for functional analysis).

Good luck with your experiments!!!!

Yes, as said XD Sha, I think that lentiviruses are the best choice, once had the constructs, making the virus and infect your cell line is very fast and not very expensive, of course, if the lab is equipped safely as said Nicolas

If you need long-term silencing shRNA is the most common way to go. If you start with siRNA and change to shRNA you are also introducing additional variables. These changes could be subtle, but since you can do both your long and short-term with shRNA you would likely be better off sticking with it. This will probably also save you time and effort optimizing for only one system.

You would also very likely have to change your targeting sequence. Probably because of the high concentration of siRNA in the cell and how they are processed you can often get knock down with and siRNA, but expressing the same target sequence from a vector may not give you significant knockdown.

You can make stable lines with virus or plasmid as long as you have a selection marker. The efficiency is much higher with virus though. Virus is efficient enough that some people freeze back the whole population of transduced cells rather than going through clonal selection. This is works in many cases, but you can get population drift from a heterogeneous population.

I am the product manager for transOMIC technologies. We have a retroviral shRNA vector that can be used to transfect as plasmid or produce virus to transduce. We have a collaboration with Cold Spring Harbor Laboratory where Greg Hannon and colleagues functionally screened over 250,000 hairpins to develop a new shRNA design algorithm. Now we are the only company that guarantees all of their hairpins will function. So, this could save you some time optimizing and trying to find shRNA that work.

We have also recently done some work with Clontech to validate their packaging and concentration system with our products. Their kits are great and will make your life much easier especially since you have not produced virus before. Then with their concentrator kit you don't even need an ultracentrifuge. You just mix the reagents, put it in the refrigerator then spin in a normal centrifuge.

In the US you need a BSL2 lab to produce retroviral particles. You should see what the requirements are for your institution.

So, I think your most straightforward path ahead with the fewest variables would be vector based RNAi transfected as plasmid then, if needed, combined with a kit for packaging and concentration. From your description, I would be surprised if our collection did not meet your needs. If you have any specific questions about how it would work with your project you can email our technical support and direct the questions to me or post here.

If your long-term knockdown experiments depend on results from the short-term experiment then you could just transfect your cells for the short-term experiment while setting up to make virus for your long-term experiment.

Hope this helps.

-Andy

Couple of things that haven't been covered or only briefly:

1) I can't stress as much as needs to be how important a good (or several) negative controls are. Scrambled controls can cause tons of side effects including being immunoactive. So I always recommend using "established" negative controls which usually are sequences targeting for example GFP and luciferase and have been published.

2) siRNAs are having very (!) different ... rules of engagement ... compared to shRNAs, the loop plays a rule, transformation of si into shRNA and still be active is still rather Voodoo (aka: a game of luck often) though the loop of hsa-miR30 seems to be not too bad. That is even more the case for miRNA based shRNAs which come from miRNA backbones, in these cases strand selectivity (to reduce off target effects from the passenger strand being incorporated instead the intended guide) is less of an issue. Plus the promoters you use have an effect including perturbation of your miRNA endogenous pathway from overexpression and seeing all kinds of side effects from this. This is a complex field and it might be a good idea to get in contact with someone who actually specializes on this and collaborate.

3. Whatever plasmid you use, make at least sure you can either directly integrate it (low effectivity) as stable after transfection (G418 based selection or such) or it already comes in a viral backbone. For Lentivirus you need S2 clearance and an ultracentrifuge, and yes, doing it based on reading a protocol can be tricky. Retroviruses are S1 only and can be easier. It depends on your cell type what you want to transduce, do the cells divide, are they prone to immune activation.

4. A stably transduced cell line means you must understand the effect of the shRNA for good. As was mentioned, if the gene plays a role in freezing/thawing/recuperation, your cell line will die most likely when stressed. If you use miRNA-based shRNAs you can use a polymerase II promoter - for this there is for example the tetON/Off system allowing to induce the expression. That can solve these problems. Of course different promoters can have different silencing properties too, depends at least in case of CMV according to literature on the cell type too ...

Bottom line: Way too little information to make a reasonable suggestion but a much more complex topic than what you seem to assume is there. Sorry. ;)

we have some experience in Sertoli cells both in vivo and in vitro, please check:

E. González-González, P. P. López-Casas and J. del Mazo “Gene silencing by RNAi in mouse Sertoli cells”. Reproductive Biology and Endocrinology. 6:29 (BioMed Central) doi:10.1186/1477-7827-6-29 (2008). http://www.rbej.com/content/6/1/29

Can anyone help to answer "Is it just a matter of time between transient and stable transfections by shRNA?"

many thanks

Dear Francesco,

Lentivirus is really an efficient way to generate stable cell lines with any other gene ORF expression, shRNA/miRNA and CRISPR/Cas9. Integration into host cell genome, it mediates long-term and stable expression of exogenous genes with a simple system for vector manipulation and production. With broad range of host, lentivirus guarantees efficient transfection in both dividing and non-dividing cells.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.