# Tissue-specific labeling of endogenous proteins with split fluorescent proteins

> **NIH NIH R15** · UNIVERSITY OF CALIFORNIA, MERCED · 2020 · $459,500

## Abstract

PROJECT SUMMARY
Protein labeling by fusion with genetically encoded fluorescent proteins has been a powerful tool for
investigating biological processes, allowing scientists to observe and analyze protein expression, localization,
and dynamics in living cells. However, traditional approaches for expressing fluorescent fusion proteins
possess drawbacks including potential overexpression artifacts, and new methods are needed, especially for in
vivo studies.
Our lab studies vertebrate organ formation using the zebrafish (Danio rerio) model system. In zebrafish and
many other model organisms, expression of fluorescent fusion proteins is often achieved by injection of in vitro
transcribed mRNA, which provides ubiquitous expression, or by transgenesis, which utilizes gene regulatory
elements to drive spatially and/or temporally restricted expression. However, both approaches run the risk of
producing overexpression artifacts. An alternative approach is to “knock-in” fluorescent coding sequences into
the genetic locus for the protein of interest. Although this approach preserves endogenous regulation of
expression, targeted insertion can be technically difficult to achieve. Moreover, many proteins are expressed
quite broadly, and fluorescent protein tagging at the endogenous locus does not allow one to study the tissue-
specific roles of such proteins.
To overcome these limitations, we propose using a split fluorescent protein approach to achieve tissue-specific
and endogenous protein labeling. Split fluorescent proteins consist of protein fragments that are expressed
independently and possess little to no fluorescence on their own. However, when present in the same cell, the
fragments self-assemble into a fluorescent complex. In this proposal, we will use a recently developed two-
component system based on the green fluorescent protein mNeonGreen2 (split-NG). By expressing one
component of the split-NG pair under a tissue specific promoter while fusing the second component to a
protein of interest via genomic “knock-in”, our technique will enable tissue-specific examination of broadly
expressed proteins. This technique has the potential to open new lines of inquiry in many fields of biological
and biomedical research.

## Key facts

- **NIH application ID:** 10046696
- **Project number:** 1R15HD102829-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, MERCED
- **Principal Investigator:** Stephanie Woo
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $459,500
- **Award type:** 1
- **Project period:** 2020-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10046696

## Citation

> US National Institutes of Health, RePORTER application 10046696, Tissue-specific labeling of endogenous proteins with split fluorescent proteins (1R15HD102829-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10046696. Licensed CC0.

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