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Genes Cells. 2013 Jun;18(6):450-8. doi: 10.1111/gtc.12050. Epub 2013 Apr 11.

Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.

Author information

1
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan.

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.

PMID:
23573916
PMCID:
PMC4834911
DOI:
10.1111/gtc.12050
[Indexed for MEDLINE]
Free PMC Article

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