# Direct generation of complex genetically-modified mouse models via embryonic stem cells

> **NIH NIH R21** · WEILL MEDICAL COLL OF CORNELL UNIV · 2022 · $254,250

## Abstract

PROJECT SUMMARY: Genetically modified (GM) animals are essential tools for the
study of both fundamental biology and human diseases. The production of GM animals
relies on two critical technologies: 1) stable genetic modifications and 2) germline
transmission of the mutations into a model system. A typical approach for creation of
complex GM mice involves the generation of tetra-parental chimeras from normal
embryos and GM embryonic stem (ES) cells, followed by multiple rounds of breeding to
obtain both male and female mice for germline propagation. Two limitations dominate
this approach. First, maintenance of pluripotency limits the complexity of genetic
manipulations. Second, this process is time-consuming, laborious, and costly,
particularly if the final objective requires many independent germline manipulations in
the same animal. We propose a feasible strategy to accelerate the production of
complex GM mouse models. Employing the technology of sex-reversion via
CRISPR/Cas9-meditated Y chromosome deletion in male ES cells and our novel ES
cell culture system, we can directly generate isogenic male and female mice from the
same targeted ES cells through tetraploid complementation (4n). This strategy would
bypass at least two mouse breeding generations: the chimera development step and
the complex breeding process. We will target male (XY) ES cell lines for intended
genetic alterations and follow the deletion of Y chromosome to generate monosomic XO
female ES cells. Using this strategy, compound homozygous GM mouse strains could
be established at unparalleled speed and costs. In this R21 application we propose the
following two Aims: Aim 1: Optimize AX-based ES cell culture system for the production
of GM mouse models. Aim 2. Direct generation of isogenic male and female complex
GM mice using novel ES cell culture medium. If successful, our approach would have a
great impact on GM mouse model construction in terms of versatility, speed, and cost.
This ambitious endeavor to develop a breakthrough technology for creation of complex
GM mouse models would also possibly foster novel research opportunities.

## Key facts

- **NIH application ID:** 10354630
- **Project number:** 1R21OD031973-01
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Duancheng Wen
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $254,250
- **Award type:** 1
- **Project period:** 2022-03-15 → 2024-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10354630

## Citation

> US National Institutes of Health, RePORTER application 10354630, Direct generation of complex genetically-modified mouse models via embryonic stem cells (1R21OD031973-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10354630. Licensed CC0.

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