Imaging-Based In Situ Analysis of 5-Methylcytosine at Low Repetitive Single Gene Loci with Transcription-Activator-Like Effector Probes.

ABSTRACT: Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins that can be used for sequence-specific, imaging-based analysis of cellular 5-methylcytosine. However, this has so far been limited to highly repetitive satellite DNA. To expand this approach to the analysis of coding single gene loci, we here explore a number of signal amplification strategies for increasing imaging sensitivity with TALEs. We develop a straightforward amplification protocol and employ it to target the MUC4 gene, which features only a small cluster of repeat sequences. This offers high sensitivity imaging of MUC4, and in costaining experiments with pairs of one TALE selective for unmethylated cytosine and one universal control TALE enables analyzing methylation changes in the target independently of changes in target accessibility. These advancements offer prospects for 5-methylcytosine analysis at coding, nonrepetitive gene loci by the use of designed TALE probe collections.

SUBMITTER: Jung A 

PROVIDER: S-EPMC9942090 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature