Related Experiment Video
Updated: May 13, 2026
06:21
An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness
Published on: May 7, 2018
A double-switch vector system positively regulates transgene expression by endogenous microRNA expression (miR-ON
Mario Amendola1, Alice Giustacchini, Bernhard Gentner
1Division of Regenerative Medicine, Gene Therapy and Stem Cells, San Raffaele Scientific Institute, Milan, Italy.
Molecular Therapy : the Journal of the American Society of Gene Therapy
|February 27, 2013
Summary
Researchers developed a novel lentiviral vector system to directly report microRNA (miR) activity. This system enables live monitoring and selection of miR-expressing cells, advancing the understanding of gene regulation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biotechnology
Background:
- Precise characterization of microRNA (miR) expression is crucial for understanding development and disease.
- Previous lentiviral vector (LV) systems reported miR activity via transgene suppression, hindering direct cell identification.
- A method is needed to couple transgene expression directly to miR activity for positive reporting.
Purpose of the Study:
- To develop a novel LV-based system for direct, positive reporting of miR activity.
- To enable live monitoring and selection of cells based on specific miR expression.
- To advance the study of miR regulation in biological processes.
Main Methods:
- Engineered LV encoding two in-series OFF switches: a miR-target-tagged transcriptional repressor and a repressor-controlled reporter cassette.
- Reporter expression is activated only when miR represses the transcriptional repressor.
- Tested the system with different repressors, vectors, and delivery methods in human primary hematopoietic stem/progenitor cells.
Main Results:
- Successfully coupled transgene expression to miR activity for positive reporting.
- Demonstrated live monitoring of two miRs during human hematopoietic stem/progenitor cell differentiation in vivo.
- Validated the system's performance across various repressors and vector configurations.
Conclusions:
- The developed LV system provides a powerful tool for direct, live monitoring and selection of miR-expressing cells.
- This technology facilitates the study of miR dynamics in cellular processes like differentiation.
- Potential applications include imaging rare miR-expressing cells and targeted gene regulation.
More Related Videos
Related Concept Videos
01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...