Inducible Assay Cell Models for Functional Gene Analysis

Inducible gene expression is a valuable tool for functional gene analysis as cells adapt to genetic modifications and can lead to the selection of mutants escaping the genetic phenotype. Stable cell populations are generated with lentiviruses expressing the TET-inducible gene expression cassette. During this process no selection pressure is applied to cells related to a genetic change induced by expression or silencing of a target gene.

SIRION Biotech offers expression systems for inducible over-expression and knockdown of genes. based on the selection of active shRNAs previously identified with RNAiONE and latest 3rd generation lentivirus technology, a near quantitative inducible knockdown is even achieved in polyclonal cell pools. This makes clonal cell line selection a redundant step.

Technical Feature

SIRION Biotech has designed a novel ONE vector lentivirus system enabling the use of one instead of two lentiviruses for generation of stable cell pools with inducible gene expression. This lowers the risk of genetic alterations which may occur during the initial step of stable cell pool generation. Moreover, advanced 3rd generation technology enables highly homogeneous polyclonal cell pools making time consuming clonal selection a redundant step.

SIRION Biotech developed

  • a ONE vector TET-ON/ TET-OFF inducible system
  • GOI/shRNA and TET-responsive transactivator are expressed from the same vector
  • Since cloning capacity is limited to 4.5 kb, for larger constructs the TWO vector system can be used where the TET responsive transactivator is expressed from an additional vector
  • Both vectors are highly sensitive to Doxycycline induction without leakiness in the absence of Doxycycline
  • In case of gene silencing, shRNA expression occurs within a microRNA backbone driven by a RNA Polymerase II promoter

Inducible stable cell pools are highly homogeneous:

Application Example

Generation of inducible overexpression cell pools:

Fadu cells stably transduced with lentiviral vector for inducible expression of RFP when grown for 48h in medium supplied with or without 1,0 µg/ml Doxycycline.

Generation of inducible knockdown cell pools:

For the purpose of target validation of a novel oncology target GOIx, an inducible gene knockdown of GOIx was induced in human A549 cells.

After selection of an active shRNA sequence against GOIx using the RNAiONETM selection platform the best sequence was cloned into the 3G All-In-One lentivirus vector followed by lentiviral transduction and stable cell pool selection. While already the stable cell pool displayed an inducible knockdown of overall 90% on mRNA, the subsequent clonal selection confirmed homogeneity and even led to the identification of cell clones with near quantitative inducible gene knockdown.