Deborah Vassar

Abstract 4895: Tagging endogenous genes with fluorescent reporters using CompoZr® zinc finger nuclease technology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks (DSBs) in DNA at desired locations. DSBs are important for site-specific mutagenesis in that they stimulate the cells natural DNA-repair processes, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The formation of a DSB increases the rate of HR by a thousand fold between a specific genomic target and a donor plasmid in somatic cells. Sigma-Aldrich CompoZr® ZFN technology is a useful tool to create site specific mutations, and to generate knock-in and knockout model lines for academic and clinical research. By this approach, we tagged an oncogene, several cytoskeletal genes and a chromatin gene by integrating a fluorescent protein sequence into the desired location (at the sequences encoding either N-terminus or C-terminus of the target protein) in the genome. The integration resulted in endogenous expression of the corresponding fusion proteins (green, red, or blue) that shows their native characteristic pattern. The following five loci were tagged: ERBB2/HER2 (human epidermal growth factor receptor 2, plasma membrane), TUBA1B (α-tubulin 1b, microtubule), ACTB (β-actin, actin stress fibers), LMNB1 (lamin B1, nuclear envelope) and HMGA1 (high mobility group AT-hook 1, nucleus). Single cell clones were isolated in SKOV3, U2OS, and MCF10 cells with one copy of a given gene tagged. Multiplexing was demonstrated by labeling different genes in the same cell line as well as different alleles of the same gene. We demonstrate that ZFN mediated gene tagging is an efficient way to generate knock-in cell lines to report endogenous gene expression. It provides the basis for development of various high content screening (HCS) assays for compound screening where target gene regulation and corresponding protein function are preserved. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2011-4895