# A CRISPR-based modular transgenic system to advance in vivo investigations of angiogenesis and fibrosis

> **NIH NIH R21** · UNIVERSITY OF VIRGINIA · 2022 · $242,250

## Abstract

Transgenic lineage-tracing mice are among the most influential biomedical research technologies. Dual
lineage-tracing mice provide further information on phenotypic switching of cell fates during normal
development, organ function, and disease. This proposal seeks to generate a series of new unique dual
lineage-tracing transgenic mice to investigate angiogenesis and fibrosis in multiple disease models.
Cycling endothelial cell (EC) are required for angiogenesis and endothelial-to-mesenchymal transition
(EndMT) participates in fibrosis of disease. However, delineating the contributions of adult ECs that
reenter the cell cycle or undergo EndMT remains a challenge. Although current lineage-tracing mice label
cells, these approaches cannot ablate specific subpopulations of ECs that cycle or undergo EndMT to
determine their contributions to disease progression and severity. To address this knowledge gap, we
will use our previously published methods of sequential orthologous DNA recombinases to fine-tune DNA
recombination temporally in specific cells. Sequential DNA recombinases allow the ablation and
interrogation of subsequent effects on organ function. Applying this strategy to cardiomyocytes (CMs),
we created a new transgenic mouse that expressed tandem orthologous DNA recombinases. We
observed that ablating endogenous cycling CMs worsened heart function after myocardial infarction (MI).
We used conventional mouse transgenesis for the CM experiments, a laborious, expensive, and time-
consuming process, taking over twelve months to obtain mice needed for investigations. However,
advances in “Targeted Integration with Linearized dsDNA” (TILD) - “Clustered Regularly Interspaced
Short Palindromic Repeats” (CRISPR) provide a remarkably accurate, efficient, and rapid alternative to
generate transgenic mice. Therefore, we will merge CRISPR-mediated genome editing and orthologous
DNA recombinases to create a toolbox of next-generation transgenic mice that can be “mixed-and-
matched” to investigate EC cycling and EndMT-mediated fibrosis in vivo. The new mice will expand our
knowledge of EC proliferation and EndMT-mediated fibrosis in multiple diseases, including cardiac
fibrosis, atherosclerosis, pulmonary arterial hypertension, cirrhosis, and cancer. The methods developed
will enable the rapid creation of various transgenic mice that use sequential DNA recombinases to restrict
Cre expression and investigate cell cycling and cell fate switching in vivo.

## Key facts

- **NIH application ID:** 10408193
- **Project number:** 1R21OD032144-01
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Matthew J Wolf
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $242,250
- **Award type:** 1
- **Project period:** 2022-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10408193

## Citation

> US National Institutes of Health, RePORTER application 10408193, A CRISPR-based modular transgenic system to advance in vivo investigations of angiogenesis and fibrosis (1R21OD032144-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10408193. Licensed CC0.

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