Identify and Validate the Function of Biological Targets
The advent of fast and reliable next generation technologies for the genomic and proteomic characterization of biological specimens has contributed to the identification of a wealth of targets that can be potentially exploited for treating cancer. However an in-depth molecular understanding of cellular processes is required to transition potential targets into pharmacological candidates.
At CrownBio, we use a comprehensive tool set to determine the biological functions of each target as well as the consequences of pharmacological intervention.
In Vitro, In Vivo, and Ex Vivo techniques
The specific knockdown of a gene of interest by RNA interference (RNAi) is a powerful tool for target discovery or validation. Gene knockdown is achieved through the use of small interfering RNAs (siRNAs) for transient downregulation of the gene of interest in the cell line.
We have expertise using a wide variety of lipid-based transfection reagents to deliver siRNAs to the cells of interest. Our robust systems and processes ensure that expression of the target gene of choice and the protein it encodes is significantly reduced.
Gene Cloning and Stable Transfection
De novo or enhanced expression of genes in mammalian cells can be achieved through molecular cloning, with genes of interest cloned and overexpressed in the cells of choice for target validation.
We utilize a variety of expression systems (both plasmid- and lentiviral-based) to deliver constitutive or inducible high level expression of wild type and mutated genes (e.g. constitutively active or kinase dead mutants).
Following establishment of stable recombinant cell lines, downstream effects of gene overexpression can be monitored through in vitro or in vivo assays.
Real-time PCR (RT-PCR) provides a quantitative comparison of gene expression in cell lines or tissues, and is used to determine whether different gene expression levels are associated with a particular cell phenotype. At CrownBio we also use RT-PCR in conjunction with RNAi technology to verify changes in gene expression.
Target Protein Knockdown and Overexpression
A variety of assays, including western blot analysis, flow cytometry, immunohistochemistry, and immunofluorescence are routinely used at CrownBio to determine knockdown or overexpression of the target protein of choice.
In Vivo/Ex Vivo Target Validation
Thanks to our unique collection of validated and well-characterized Cell Line Derived Xenograft tumor models, human primary cancer cells (PrimePanel™), and patient tumor samples we can correlate in vitro preclinical findings with in vivo data, which can be translated to clinical patient outcomes.
The effect of interfering with a gene function can be evaluated in vivo using tumors grown in mice as xenografts or ex vivo using tumor tissue or tumor derived cell suspensions. The activity of your targeted agent on tumor growth can be measured directly and target-specific biomarkers can be investigated using our unique models.
Reporter Gene Assays
Reporter assays enable monitoring of enhanced expression or nuclear localization of transcription factors. Binding of the transcription factor enables transcription and translation of the reporter gene, which can be quantified in vitro or in vivo by measuring light emitted in the presence of the substrate.
We provide a number of reporter gene assays to measure specific conditions in vitro or in vivo (e.g. hypoxia) in real time, or to follow tumor growth and metastasis over time.
Contact us today for expert advice as to how we can help validate your target.