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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Published on: January 6, 2015

Fluorescent protein-assisted purification for gene expression profiling.

M Raza Zaidi¹, Chi-Ping Day, Glenn Merlino

¹Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)

|December 1, 2010

Summary

This summary is machine-generated.

Researchers developed methods to purify rare cells, including skin stem cells and cancer cells from lung metastases, using fluorescent proteins and flow cytometry. This enables in vivo tracking and gene expression profiling for molecular analysis.

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Area of Science:

Cellular and Molecular Biology
Biotechnology
Cancer Research

Background:

Flow cytometry with fluorescent proteins enables purification of rare cells from complex tissues.
This technique is valuable for molecular analysis of cancer cells, separating them from tumor stroma.
Bioluminescence and fluorescence allow in vivo tracking of cancer cells.

Purpose of the Study:

To describe protocols for purifying rare skin stem cells.
To detail methods for in vivo monitoring and purification of cancer cells from lung metastases.
To outline RNA isolation from purified cells for gene expression profiling.

Main Methods:

Utilizing cell type-specific expression of fluorescent proteins for cell purification.
Employing flow cytometry for isolating specific cell populations.
Coexpressing bioluminescence and fluorescence for in vivo tracking and subsequent purification.
Isolating total RNA from purified cells for gene expression analysis.

Main Results:

Successful purification protocols for rare skin stem cells were established.
In vivo monitoring and purification of cancer cells from lung metastases were demonstrated.
Protocols for RNA isolation and gene expression profiling from purified cells are presented.

Conclusions:

Fluorescent protein-based cell purification is effective for rare cell isolation and molecular analysis.
The described methods facilitate in vivo tracking and purification of cancer cells at various tumorigenic stages.
This approach supports comprehensive gene expression profiling of purified rare and cancer cells.