title: 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.
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.
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Arildo
authors.initials: A
authors.lastname: Nerys-Junior
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Lendel C
authors.initials: LC
authors.lastname: Costa
authors.affiliation: Plataforma de Animais Transgênicos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil .
authors.forename: Luciene P
authors.initials: LP
authors.lastname: Braga-Dias
authors.affiliation: Laboratório de Pesquisas Sobre o Timo, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil .
authors.forename: Márcia
authors.initials: M
authors.lastname: Oliveira
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Atila D
authors.initials: AD
authors.lastname: Rossi
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Rodrigo Delvecchio
authors.initials: RD
authors.lastname: da Cunha
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Gabriel S
authors.initials: GS
authors.lastname: Gonçalves
authors.affiliation: Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil .
authors.forename: Amilcar
authors.initials: A
authors.lastname: Tanuri
keywords: CCR5; HMA; TALEN; gene knockout; genome editing