# The development of an assay to study chromosome fragility in human cells

> **NIH NIH UH2** · GEORGIA INSTITUTE OF TECHNOLOGY · 2020 · $186,136

## Abstract

Gross chromosomal rearrangements (GCRs) including chromosome translocations, deletions, inversions, and
amplifications are hallmarks and proven sources of many tumors and hereditary diseases in humans.
Identification of exogenous and endogenous factors that induce GCRs and underlying mechanisms of
chromosome instability are important for our understanding disease prognosis and strategizing approaches for
disease prevention and treatment. There are few assays that monitor chromosome instability in human cells
and to our knowledge none of them can address complex rearrangements frequently detected in cancers.
Moreover, GCRs are often studied in cancer cell lines that have dysregulated cellular metabolism and altered
karyotypes, which greatly complicates the analysis of mechanisms of genome instability. The objectives of the
proposed research are i) to develop a sensitive assay for detecting different types of chromosomal instability in
non-cancerous human cells and ii) to characterize the effect of endogenous and exogenous damaging agents
on the induction of GCRs. Specific Aim 1 is to introduce an engineered GCR cassette into two primary
immortalized non-cancerous and karyotypically stable male cell lines. We will modify subtelomeric regions of
chromosome X in BJ-hTERT fibroblasts and RPTEC/TERT1 renal epithelial cells using a CRISPR/Cas9
system. The GCR assay is based on the use of fluorescent markers well as gene dosage folA marker. This
system allows to monitor chromosome X deletions and gene amplification as well as associated
rearrangements. Specific Aim 2 is reveal the effect of DNA fragile motifs on the induction of GCRs. The
system provides the opportunity to target any sequence motif along with the GCR cassette to the same
location on chromosome X. Therefore, any DNA sequence that is suspected of causing double-strand breaks
(DSBs) and triggering genome instability can be assessed for its potential to induce deletions and gene
amplification in the chromosomal context. We will determine the effect of palindromic sequences and
GAA/TTC tracts, which were shown to induce DSBs and chromosomal rearrangements in yeast, on the
induction of GCRs in human cells. The resulting genome rearrangements will be analyzed using chromosome
spreads coupled with fluorescent in situ hybridization and comparative genome hybridization. siRNA- or
CRISPR/Cas9-mediated knockdown will be carried out for genes that have been shown to affect the repeat
metabolism or repair processes. Specific Aim 3 is to study the effect of exogenous damage on the induction of
GCRs. Cell lines that are under development can be used to determine the effect of any exogenous factors on
their ability to trigger GCRs. We will evaluate the frequency of GCRs upon treatment of cells with established
DNA-damaging agents such as ionizing radiation, UV-light, methyl methanesulfonate and etoposide. Overall,
this unique experimental system will be instrumental in dissecting mechanisms of chromosoma...

## Key facts

- **NIH application ID:** 9962458
- **Project number:** 5UH2GM129516-02
- **Recipient organization:** GEORGIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** KIRILL S LOBACHEV
- **Activity code:** UH2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $186,136
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9962458

## Citation

> US National Institutes of Health, RePORTER application 9962458, The development of an assay to study chromosome fragility in human cells (5UH2GM129516-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9962458. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*