Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.
Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.
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DOI：
10.1111/gtc.12050
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发表时间：
2013-06
期刊：
影响因子：
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通讯作者：
Kawahara A
中科院分区：
文献类型：
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作者：
Ota S;Hisano Y;Muraki M;Hoshijima K;Dahlem TJ;Grunwald DJ;Okada Y;Kawahara A
异源双链泳动试验（HMA）广泛用于表征人类病毒的毒株变体。为了确定它是否可以检测同源模板中的小序列差异，我们在pBluescript II的多克隆位点（MCS）中构建了一系列缺失构建体（1-10 bp缺失）。使用野生型和缺失构建体之一的混合物对MCS进行PCR扩增后，使用15%聚丙烯酰胺凝胶对所得PCR扩增子进行电泳。两种类型的heteroduplexes表现出延迟电泳迁移相比，个别homoduplexes。因此，我们应用此HMA检测转录激活因子样效应核酸酶（TALEN）诱导的插入和/或缺失（indel）突变的内源性基因座。我们发现，TALEN体内活性很容易通过来自TALEN注射的F0胚胎的多个HMA谱的程度来估计。此外，TALEN注射的F0代创始人鱼在F1代胚胎中产生了几个独特的HMA谱。序列分析证实，不同的HMA谱含有不同的indel突变。因此，HMA是用于检测TALEN介导的基因组修饰的快速且灵敏的分析方法。
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.