Evaluating the morphology and growth rate of CRISPR clones with edited ITS regions
Like the plastic end of a shoelace, telomeres act as “caps” that protect the end of chromosomes to maintain chromosomal stability and prevent chromosomal degradation. As the cell divides, telomeres get shorter, providing a correlation between aging and telomere length. In addition, telomeres are thought to interact with distant regions of the chromosome is via telomere position effect over long distance (TPE-OLD) to modulate gene expression. Only a few specific telomeric chromatin interactions have been reported. The Barthel Lab conducted a screen to identify telomere-chromatin interactions and identified ITS regions as potential interaction partners. Furthermore, to elucidate the biological functions of these ITS regions, a CRISPR strategy was used to generate ITS knockout clones in a fibroblast cell line, BJhTERT. The current study analyzes the nuclear morphology and growth rates of these knockout clones. Fluorescence microscopy with DAPI staining was used to screen the ITS knockout (KO) clones for the nuclear abnormalities of micronuclei or multilobed nuclei. Morphological analysis revealed a low incidence of micronuclei but a higher prevalence of multilobed nuclei (30-40%) in ITS KO clones compared to the BJ hTERT parental cell line. To measure growth rates, cells were cultured for 10-14 days and then passaged and counted. The growth rate data indicates that ITS KO clones have slower growth in comparison to BJ hTERT cells. These findings suggest that the ITS region plays a role in influencing nuclear architecture and in maintaining normal cell growth.
