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Pathway Profiling via Endogenous Gene Tagging

Thu, 02/14/2013 - 1:54pm
Dmitry Malkov, PhD, Principal Scientist; David Briner, Manager, Emerging Technologies; Sigma Life Science, St. Louis, Mo.
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Figure 1. Targeted integration strategy at the EGFR c-terminus. (All figures: Sigma Life Science)

Since the isolation and propagation of the first immortalized cell line some 60 years ago, a multitude of relevant cell types and lineages now serve as a cornerstone in scientific research. However, as the “toolbox” grows so does our understanding of potential shortcomings in these models.

Overexpression can provide powerful insights into elucidating gene function. However, they sometimes provide an inaccurate picture due to lack of appropriate context in the cell, or fail to adequately control for an isogenic setting. Recent studies have demonstrated an acute need to study protein localization and function in the context of the true endogenous locus.1 Such findings prodded scientists at Sigma Life Science to fluorescently tag the endogenous epidermal growth factor receptor (EGFR) locus in A549 cells to create a more complete tool for studying EGFR.

EGFR belongs to a class of receptor tyrosine kinases (RTKs). Abnormally high expression of EGFR has been correlated with many types of cancer. Overexpression of EGFR and other RTKs leads to constitutive activation of oncogenes, thus qualifying them as high-profile cancer targets. Unfortunately, a high rate of EGFR mutagenesis in cancer results in a loss of efficacy for drugs inhibiting its kinase activity.2

Current EGFR activation and internalization assays are based on overexpression of EGFR-GFP fusion protein or detection of phosphorylated EGFR by immunohistochemistry. A major limitation of these methods is their inability to detect EGFR in living cells. To fill this gap, Sigma scientists developed an A549 cell line that is modified at the endogenous EGFR locus to report activation and localization of the protein. Notably, this provides for visualization of native EGFR expression and localization in live cells. Most traditional gene targeting methods in mammalian cells utilize plasmid-based constructs with large autologous arms to facilitate homologous recombination. These methods usually require an extensive screening effort due to low efficiencies and can result in non-specific integration of the targeting construct. A more efficient strategy to generate specific modifications emerged from the development of zinc finger nucleases (ZFNs), which create a double-strand break at a targeted locus and boost homologous recombination rates from 1,000- to 10,000-fold.3

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Figure 2: Expression and localization of endogenously-tagged EGFR.

Using CompoZr ZFNs in conjunction with a targeting construct, a fluorescent transgene was inserted into the C-terminus of EGFR (Figure 1). The resulting fluorescent fusion protein was stably and specifically integrated into the cells and expressed under endogenous regulation of gene expression and protein function.

Using a tagged protein under endogenous regulation allows detection of RTK activation in live cells. Activation of EGFR leads to receptor internalization in wild type cells. Presence of the fluorescent tag on endogenous EGFR allows tracking of this normal internalization process after the addition of EGF to the cells (Figure 2). Internalization kinetics can then be characterized by quantifying the fluorescent signal through software-driven imaging. Application of a selective inhibitor of EGFR, Tyrphostin AG 1478, verifies target-dependent effect.

Sigma has created a panel of cell lines with tagged cytoskeletal and pathway marker genes at disease-relevant loci aid in characterizing function and potential drug interactions. A variety of breast cancer and oncology gene knockout cell lines have been created in many relevant cell types. More recently Sigma scientists have tagged genes in human induced pluripotent stem cells for reporting expression of cardiac, neuronal, and hematopoietic markers once the cells differentiate into their respective lineages.

Targeted gene editing allows one to study biology in a more “native” context, without having to concede to potential limitations inherent in traditional methods.

References
1. Doyon JB, et al. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat Cell Biol. 2011;13(3):331-7.
2. Quesnelle KM, Grandis JR. Dual kinase inhibition of EGFR and HER2 overcomes resistance to cetuximab in a novel in vivo model of acquired cetuximab resistance. Clin Cancer Res. 2011; 17:5935–44.
3. Elliott B, et al. Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol. 1998;18(1):93-101.

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