SIRION Biotech - frequently asked questions
My lab is restricted to BSL 1. How can I benefit from the RNAiONETM system / SIRION’s viral vector portfolio?
SIRION Biotech offers custom cell pool generation services for their lentivirus vector systems. The finished cell pool will be free of any residual virus particles, making it safe for biosafety-level 1.
How homogenous is the finished knockdown cell pool? Will it require clonal selection?
Our cell pools are highly homogenous (>95% transduction positive). This makes clonal selection obsolete for most applications.
My target is involved in cell cycle or growth processes. I suspect that knockdown might inhibit cell growth/survival. Can I still use the RNAiONETM system?
We offer an inducible system for these purposes. This system enables the genomic integration of the validated shRNA in a dormant state, making standard proliferation of the manipulated cell pool possible. The shRNA expression can then be triggered at any time by application of doxycycline.
Can I purchase the RNAiONETM service for any gene?
We reserve the right to run a test of feasability with the gene sequence before we decide on guaranteeing any success. If we can’t give a guarantee the client can withdraw from the project at no cost.
The shRNA validated by RNAiONETM worked well, but now I want to switch from a constitutive expression system to an inducible system. Can I use the same shRNA again?
Our shRNA validation system is constructed to simulate the end application (constitutive vs. inducible) of the shRNA sequence. The validation platform varies in key elements, depending on what the future application is. Translation from one platform to another can lead to reductions in knockdown efficiency. To avoid this, we highly recommend testing the shRNA and several alternatives in the new expression environment through another RNAiONETM trial. We offer special services at a smaller scale for this purpose.
I made my own vector system using your validated shRNA information but only get low knockdown. How is this possible?
Our shRNA validation system is constructed to simulate the end application (constitutive vs. inducible) of the shRNA sequence. The validation platform varies in key elements, depending on what the future application is. Translation from one platform to another can lead to reductions in knockdown efficiency. Another source for error could be low transduction rates.
Does SIRION provide production of high titers beyond the listed quantities?
Higher titers can be possible depending on the specifics of the vector construct. Please contact a representative to discuss this detail in person.
What are the gene size limitations for the viral vector construction?
Adenovirus: 7.5 kb
Lentivirus: 5 kb constitutive/ 4 kb inducible
AAV: 4,5 kb
Listed are the total capacity limitations. Promotor- and reporter-systems are not included in these numbers. Please contact a SIRION Biotech representative to discuss the details of your project.
Are cells treated with lentiviral vectors free from residual virus particles?
All cells pools are tested for virus retention before they are shipped to make absolutely sure they adhere S1 safety standards.
Can I use cells or virus particles modified by SIRION Biotech commercially?
SIRION Biotech services are offered for preclinical applications for research use only. None of the products made by SIRION may be used for commercial applications unless this has been discussed otherwise. Should you be interested in commercial applications please contact SIRION Biotech for possible co-development/-marketing strategies.
Major disadvantages of RNAi technology are OFF target effects.
SIRION`s RNAiONE service identifies shRNA sequences for near quantitative knockdown, minimizing the risk to work on OFF target effects. Additionally, SIRION offers its VariCHECK cell model to reverse shRNA induced phenotypes by the overexpression of the undegradable WT protein sequence. VariCHECK can be implemented as additional RNAi ON target control. SIRION also recommends working with more than 1 shRNA sequence against 1 target.
The TET inducible system is not the best choice as a double positive cell pool has to be generated.
This increases the risk of site specific mutagenesis.
SIRION offers an All-in ONE inducible system. The transactivator and inducible shRNA is expressed from the same vector backbone. Only one transduction and antibiotic selection step is needed for stable inducible cell model generation.