Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Arildo Nerys-Junior; Lendel C Costa; Luciene P Braga-Dias; Márcia Oliveira; Atila D Rossi; Rodrigo Delvecchio da Cunha; Gabriel S Gonçalves; Amilcar Tanuri

doi:10.1590/s1415-47572014000100018

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Genet Mol Biol. 2014 Mar;37(1):120-6. doi: 10.1590/s1415-47572014000100018. Epub 2013 Feb 28.

Authors

Arildo Nerys-Junior¹, Lendel C Costa¹, Luciene P Braga-Dias², Márcia Oliveira³, Atila D Rossi¹, Rodrigo Delvecchio da Cunha¹, Gabriel S Gonçalves¹, Amilcar Tanuri¹

Affiliations

¹ Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
² Plataforma de Animais Transgênicos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil .
³ Laboratório de Pesquisas Sobre o Timo, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil .

Abstract

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

Keywords: CCR5; HMA; TALEN; gene knockout; genome editing.