title: Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.
abstract: The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1-10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.
authors.affiliation: Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ota
authors.forename: Yu
authors.initials: Y
authors.lastname: Hisano
authors.forename: Michiko
authors.initials: M
authors.lastname: Muraki
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Hoshijima
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Dahlem
authors.forename: David J
authors.initials: DJ
authors.lastname: Grunwald
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okada
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kawahara
mesh_terms: D000818:Animals; D003851:Deoxyribonucleases / Q000378:metabolism*; D016678:Genome / Q000235:genetics*; D020180:Heteroduplex Analysis; D054643:INDEL Mutation / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases