Transcription activator-like effector (TALE) technologies are being used in a variety of creative ways by swapping functional protein domains that are connected to the ‘programmed’ DNA binding domain. Miyanari (2014) and Miyanari et al (2013) describe their use of TALEs to visualize changes in chromatin and nuclear positioning of specific genomic sequences.
Nuclear architecture is dynamic and non-random, showing affects on gene regulation and misregulation.
DNA fluorescent in situ hybridization (FISH) is a popular method for visualizing the location of specific DNA sequences on chromosomes or within nuclei. While powerful, the method must be to fixed samples, precluding its use in visualizing living cells.
Vital labeling of DNA sequences can allow for visualization of specific DNA sequences in vivo and this can be helpful and insightful.
Miyanari et al (2013) describe their design and construction of a sequence-specific TALE fused to the fluorescent protein mClover. Miyanari (2014) provides a how-to technical guide to the use of this technology, which is referred to as TGV, TALE-mediated genome visualization.
While the TGV results are impressive as these embedded videos from Miyanari et al (2013) show, the technology, at the moment, is limited to repetitive sequences including centromeres and telomeres (lots of binding sites – lots of signal).
Serial section of confocal images of mitotic ES cells expressing TALE-mClover (green) and TALE-mRuby2 (red) against MajSat and MinSat sequences, respectively. DNA was stained with DAPI (gray).
Time lapse imaging of ES cell-line (E142C-05) stably expressing TALE-mClover against MajSat (green) and histone H2B-tdiRFP (red). Images were acquired every 3 min over 200 time frames (10 hrs).
They were also able to differentially label parental chromosomes and one SNP within a 15-nt target sequence allowed for discrimination between parental alleles.
Can TGV be used to visualize single-copy genes?
Not when using only one TALE against the target gene. So, single-copy sequence resolution must wait for improved technologies (e.g.better fluorophors)
In the mean time live visualization of microsatellites or other repeated sequences using TGV could be useful.
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