 VariCHECK inducible expression systems for functional analysis of protein variantsThe availability of cell based in vitro systems allowing for mutational analysis of protein function are increasingly important in many fields of current medical and pharmaceutical research. Specifically, when you wish to better understand cellular processes associated with disease-causing mutations in protein kinase domains, growth receptors, oncogenes and other key regulatory proteins. Or when drug resistance caused by mutations in drug interacting proteins are of interest. SIRION BIOTECH developed a next generation inducible expression system VariCHECKTM which allows for functional analysis of protein variants. VariCHECKTM is taking advantage of the lentiviral TET-inducible expression system, thus preventing for cell adaption phenotypes. VariCHECKTM enables TET-inducible knockdown of wild type gene of interest (GOIendo) with simultaneous TET-inducible overexpression of a mutant variant of the same gene (GOIecto). Technical FeatureThe novel one vector lentivirus system allows for simultaneous expression of the transgene and the reverse TET-dependent transactivator (rtTA3). Thus, tightly controlled cell pools for inducible transgene expression can be generated in only a few weeks. Being partner of TET Systems, SIRION BIOTECH is able to use latest 3G TET inducible gene expression systems to guarantee highest possible quality. VariCHECK cells are stably transduced with the following 2 lentiviral vectors.Vector 1: In the presence of doxycycline (Dox) a highly efficient shRNA sequence for near quantitative knockdown of the wild type form of your gene of interest (GOIendo) is expressed from this vector. A puromycin resistance gene together with the rtTA3 gene are expressed from a strong constitutive promoter. Vector 2: In the presence of doxycycline a mutant variant of your GOI (GOIecto) is expressed. This gene is also altered within the shRNA targeting region. Thus, no shRNA-binding and hence silencing of the GOIecto mRNA can occur. In vector 2 the constitutive promoter drives expression of rtTA3 and Neomycin phosphotransferase.
Mode of ActionCells express only the wild type form of your GOI in the absence of Dox.  Following Dox addition silencing of the WT gene and simultaneous expression of the mutant variant occurs. Thus, with VariCHECKTM cells wild type and mutant variants of your GOI are expressed in an alternating and Dox-dependent manner.
 Application ExampleMutational analysis of an oncology target (GOIx) in A549 cells
Experimental Procedure: Step 1: Identification of highly effective shRNA sequences using RNAiONETM Timeline: 2-3 weeks Step 2: Cloning of the best two shRNA sequences into lentiviral vector 1 Timeline: 2 weeks Step 3: Generation of stable cell pools Timeline: 3 weeks Step 4: Quantification of TET-inducible gene knockdown for each cell pool Timeline: 1 week Step 5: Clonal selection by limited dilution using the cell pool that mediated the best knockdown. Timeline: 5 weeks Step 6: Identification of a cell clone with near quantitative target gene knockdown Timeline: 1 week Individual cell clones were derived by limited dilution from a stable cell pool expressing an inducible shRNA. Individual clones were analyzed for the degree of inducible gene knockdown upon addition of 1µg/ml doxycyclin to the medium. The remaining expression of the target gene mRNA to the reference parental cell (100%) is given.  Step 7: Transduction of the best performing clone (here clone 5) with lentiviral vector 2 encoding a mutant variant of the GOI and generation of a stable cell pool. Timeline: 3 weeks
ResultsqRT-PCR Analysis The cell pool generated in step 7 was further analysed as follows: Expression of the GOI in the absence and presence of Dox was quantified by qRT-PCR. Towards this purpose 2 different primer pairs specific for either the endogenous or the ectopically expressed mRNA were designed. By this means relative expression levels of wild type and mutant form of the GOI can be quantified in the non-induced and Dox-induced state.
qRT-PCR amplification curves obtained for endogenous and ectopically expressed GOIx mRNA in the presence and absence of Dox using the primer pairs described above  Western Blot Analysis Expression of the GOI was further analysed on the protein level by Western Blot. For this purpose cell extracts were prepared from Dox-treated and non-treated probes of the original clonal knockdown cell line described in Step 6 and from the VariCHECKTM A549-GOI x cells. 
No GOIx expression is detectable in Dox-treated cells in the clonal GOIx knockdown cell line. However, in Dox-treated VariCHECKTM A549-GOIx cell expression levels are comparable to those of non-treated cells
Functional Analysis Dox-induced knockdown of GOIx in A549 cells resulted in reduced cell proliferation. VariCHECKTM A549-GOI x cells that ectopically express the wild type protein like parental A549 cells showed normal growth properties.  These charts show growth curves determined for the clonal GOIx knockdown cell line and VariCHECKTM A549-GOI x(WT) in the presence and absence of Dox. Ectopic Dox-induced expression of the GOIx wild type protein fully restored proliferative properties of the cells.
ConclusionsAn almost quantitative switch from wild type to the ectopically expressed form of GOIx could be shown for VariCHECKTM A549 cells upon addition of Dox, both on mRNA and protein level. Moreover, the inhibitory effect of GOIx knockdown on cell proliferation was fully rescued by ectopic expression of the wild type GOIx protein. This demonstrates that
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