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.

Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol, the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato

“…Regions surrounding the St SSR2 target sites were amplified by PCR using the primer set 59-TGTTCTCTGACACTGTTGTAGCACT-39/59-TCGAAGCATACATACCG-GTCATCAT-39 and PrimeSTAR Max DNA polymerase (Takara Bio). Amplified products were separated on 5% polyacrylamide gels (Ota et al, 2013). Two samples (lines #271-5 and #271-19) had extra bands probably due to the formation of heteroduplexes within the target sites (Supplemental Figure 6C).…”

Zebrafish foxo3b Negatively Regulates Antiviral Response through Suppressing the Transactivity of irf3 and irf7

“…After the injected embryos were incubated at 28.5˚C for 24 h, the genomic DNA was extracted from 20 to 30 embryos by heating the embryos at 94˚C for 40 min in lysis solution (50 mM NaOH) (the reaction was terminated by adding 1 M Tris-HCl [pH 8]). The mutant detection was followed by a heteroduplex mobility assay as described previously (40). When the results were positive, the remaining embryos were raised to adulthood and treated as F 0 , which were backcrossed with the wild-type (WT) zebrafish for generating F 1 .…”

Genome Editing in Mice Using CRISPR/Cas9: Achievements and Prospects

“…Besides, the two main assays used to detect induced genomic modifications (e.g. Surveyor and T7E1 assays) lack sensitivity, and other methods are currently developed [78][79][80][81].…”

“…Besides, the two main assays used to detect induced genomic modifications (e.g. Surveyor and T7E1 assays) lack sensitivity, and other methods are currently developed [78][79][80][81].Collectively, nuclease-based technologies are revolutionizing contemporary molecular genetics, and are particularly applicable to the mouse genome. We anticipate that each of these systems will be thoroughly studied and enhanced [82], allowing researchers to take advantage of each type of nucleases according to their specificity and mechanisms of action.…”