Related Experiment Video
Updated: Mar 23, 2026
Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
Published on: September 20, 2020
Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states
Partha Samadder1, Ning Weng1, Thomas Doetschman1,2,3
1BIO5 Institute, University of Arizona, Tucson, Arizona.
Abstract:
The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry.
Insights
Researchers developed new transgenic mouse lines that use Green Fluorescent Protein (GFP) to mark specific cell populations. This innovation aids in detecting early molecular changes in diseases like cancer by highlighting rare cell subsets.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Eukaryotic organs contain complex cell type interspersions crucial for function.
- Organ dysfunction in diseases like cancer often begins with subtle changes in a small cell subset.
- Detecting early molecular disease indicators is challenging due to the abundance of normal cells.
Purpose of the Study:
- To create novel transgenic mouse models for identifying and isolating specific cell subpopulations.
- To develop a tool for early detection of molecular changes associated with oncogenesis.
- To facilitate the study of transcriptional alterations in disease states.
Main Methods:
- Generation of transgenic mouse lines expressing a nuclear-localized histone 2B-Green Fluorescent Protein (GFP) fusion protein.
- Utilized a Cre-lox system for inducible expression of GFP in specific cell populations.
- Characterized the mouse lines using microscopy, flow cytometry and sorting, and single-nucleus Reverse-Transcription polymerase chain reaction (RT-PCR).
Main Results:
- Successfully generated transgenic mouse lines where specific cells exhibit green fluorescent nuclei upon Cre recombinase activity.
- Demonstrated the utility of these lines for purifying minority cell subpopulations.
- Validated the method for analyzing transcriptional profiles of sorted single nuclei.
Conclusions:
- The developed transgenic mouse lines provide a powerful tool for isolating and analyzing specific cell populations.
- These lines are particularly valuable for studying early transcriptional changes in oncogenesis.
- This technology enhances the ability to detect subtle molecular events within complex biological systems.
More Related Videos
09:20Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease
Published on: February 1, 2022
09:34A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations
Published on: October 25, 2018
