BRCA1 HeLa SilenciX

General questions

What are the SilenciX cell lines?

A SilenciX cell line is a Ready-to-use cellular model in which a specific gene has already been silenced using RNA interference. This stable and permanent model enables access to the specific involvement of proteins in cellular models.
For example, ERCC1 HeLa SilenciX (Cat nr 01-00002) are HeLa cells in which the endonuclease ERCC1 gene (involved in DNA repair) has been knocked-down.

Discover more about this stable-silenced cellular model and recent publications on the "SilenciX Tech Infos" section.

My target is not listed in your catalogue, how can I access to the SilenciX technology?

Our specialist can edit a dedicated quote for your project. The price would depend on the target of interest and the cell type you prefer. A specific feasibility study will be performed by our lab. It takes 70-90 days for the production of our SilenciX cell lines.

What is the delivery time frame for SilenciX cell lines?

Immediate dispatch of catalogued SilenciX cell lines.
70-90 days for the production of new SilenciX cell lines.

Can I buy the vector separately?

Only the SilenciX cell lines are available, vectors can not be purchased separately.

What is the level of safety required to work with SilenciX?

Our EBV-based vector for shRNA expression contains some elements of the Epstein-Barr virus such as the EBV latent origin of replication (OriP) with its two parts (DYAD symmetry and FR region). It also expresses the nuclear retention protein EBNA-1. However, it does not confer to the transfected cells the ability to produce viral particles (unlike adenovirus or lentivirus based plasmids). Therefore, the safety level required for SilenciX cell lines is the same as for the parental cells (biosafety level 2 for HeLa cells).

Target background

How do you design the shRNAs ?

The design for shRNAs is based on an algorithm called DSIR (Designer of Small Interfering RNA) which predicts RNAi efficacy and generates highly specific shRNA sequences through powerful screening to prevent off-target effects. It is one of the best performing methods among existing algorithms (Matveeva et al., 2007).

Can I access the algorithm used for SilenciX technology?

tebu-bio has access to the full version of the algorithm DSIR under license. There is another basic version freely available online which does not include all functions such as blast-screening for off-target search for example.

Can I access the shRNA sequences?

The oligonucleotide sequences complementary to the target are provided upon delivery.

Can I get a specific target gene silenced in HeLa cells?

Any gene expressed in HeLa cells can be silenced, unless the silencing proves to be lethal to HeLa cells.

Can I have a specific target gene silenced in my own cell line?

Any Human or mouse adherent SilenciX cell line can be produced as a custom project starting from your own parental cell line. These custom SilenciX cell lines will not be included in our catalogue.

Are you able to perform double-silencing with the SilenciX technology?

As a general rule, double-silencing is not currently possible with SilenciX. It is only possible for genes with sufficient sequence homology. The same applies to knock-down targeting multiple variants and isoforms.

Silencing

What is the warranty?

All catalogue SilenciX cell lines have been validated to be knocked-down by at least 70% at the RNA level.

How can I assess the knock-down at RNA level ?

Real-time PCR enables the evaluation of the knock-down in the silenced cells compared to control SilenciX cells. We do not recommend the use of other RNA detection methods (e.g. Northern analysis or conventional PCR), as they are not quantitative.

How can I assess the knock-down at protein level?

Western-blot analysis may not be the best option as it’s often designed for high level protein expression. Make sure you do not overload your gel with an excess of protein sample. We recommend you perform a titration in order to determine the sensitivity limit of the technique. The dilutions of both primary and secondary antibodies may also be optimised.

Is my target protein silenced at 100%?

The guarantee of the Silencix technology is at least 70% knock-down of the target mRNA by qPCR validation. The level of RNA extinction observed in most of the SilenciX cell lines produced so far is above 80%, generally leading to a very low or undetectable expression level of the silenced gene. Nevertheless, silencing levels may vary from one target to another.

Is the loss of function of my protein of interest guaranteed?

The SilenciX technology allows comparative studies between the silenced cell line and the control cell line. The level of loss of function may not correlate directly to the level of silencing at RNA level. There is also a possibility that the knock-down of the target turns on natural compensation pathways. In such cases, the use of several SilenciX cell lines with specific target silencing may help understand and elucidate biological pathways.

What happens in the case of redundant genes?

A loss of function should be observed unless redundant genes take over functional recovery. Such take over may also be observed in transient silencing and is not specific to the SilenciX technology. If the redundancy is known, it can be worth making a single knock-down for each redundant gene for the sake of comparison.

Is there a possibility to switch off the silencing?

Yes, it is feasible by removal of the antibiotic used for the selection of silenced cells. The level of silencing reversal varies depending on the target gene. We advise comparing SilenciX cells to the Control SilenciX cells.

Control

What is the control for the SilenciX technology?

We supply a standard control cell line with each SilenciX cell line. The control SilenciX cell line is established with an inactive shRNA that carries two mismatches in one strand of the hairpin structure. For each catalogue SilenciX reference, the Control cell line is included in the delivery.

Can my own validated control siRNA sequence be used for making the control cell line?

Yes, we can produce it as a custom project.

How do you check the silencing of my gene?

The silencing of target genes is validated by relative qPCR and we guarantee at least 70% extinction at the mRNA level. The level of silencing at the RNA level is shown in the product specification sheets.

Technical questions

Which protocol would you recommend for cell culture?

The thawing procedure as well as the cell culture protocol are described in the User Manual you will receive with your delivery, or they can be downloaded from our web site.

Which contaminants do you test in SilenciX cells?

We check the absence of Mycoplasma and the absence of bacteria in each cell line.

Is it possible to Silence non adherent cells?

The SilenciX technology is currently not available for cells in suspension.

Is over-expression of another protein within HeLa SilenciX cells feasible ?

Yes, as long as a different antibiotic for the selection of the recombinant cells is used (meaning different from hygromycin, for HeLa SilenciX cell lines listed in the catalogue).