An Application of Zinc Finger Nuclease Technology to Create Knockout Cancer Lines
Session type: Poster / e-Poster / Silent Theatre session
Oncogenes and cellular pathways represent potentially promising targets for therapeutic compounds. Among these, apoptotic pathways are differentially expressed in different patients and cancer lines. An individual patients response to chemotherapy tends to differ depending on their unique genotype with regard to the apoptotic pathway proteins. These differences have been hard to model in isogenic settings, until the advent of the Zinc Finger Nuclease (ZFN) technology. Traditionally, cell line engineering techniques to disrupt specific genes were limited to time consuming homologous recombination and screening approaches in a limited number of cell lines. ZFN technology allows rapid and permanent disruption of specific gene loci in any cell line of choice. Sequence specific nuclease cleavage, followed by imperfect DNA repair, gives rise to permanent gene disruptions. As an application of the ZFN technology, we demonstrate the construction of genetically defined cell lines.
Two pro-apoptotic regulators, Bcl2associated X protein, BAX, and Bcl2-like protein, BAK, genes were knocked out using ZFNs in three different cell lines, A549 (human lung carcinoma), DLD-1 and SW48 (human colorectal cancer). Complete knockouts were achieved, including in A549 where the BAX gene is tetraploid. DNA sequence alterations were verified by sequencing, and the absence of BAX and BAK proteins was confirmed by Western blot.
Cells lacking both BAX and BAK are impaired in the intrinsic induction of apoptosis as demonstrated by the lack of caspase activation, compared to the wild-type counterpart. The straightforward approach coupled with the power of ZFN technology to address both diploid as well as polyploid targets, makes this an ideal tool for the generation of genetically modified cell lines with their isogenic controls.
Targeted gene editing provides an important tool for genetic manipulation that enables the study of cancer genes and creates isogenic screening tools to test the sensitivity of endogenous gene modifications to therapeutics.