Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Michael  Tyler Guinn; Damiano Coraci; Lesia Guinn; G&#225;bor Bal&#225;zsi

doi:10.3791/62109

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

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09:20 min

July 6, 2021

DOI: 10.3791/62109-v

Michael Tyler Guinn^1,2,3, Damiano Coraci*², Lesia Guinn*², Gábor Balázsi^1,2

¹Biomedical Engineering Department,Stony Brook University, ²Laufer Center for Physical and Quantitative Biology,Stony Brook University, ³Stony Brook Medical Scientist Training Program

Overview

This study presents a standardized protocol for controlling light-responsive mammalian cells through optogenetics. It aims to facilitate the implementation of light-induced gene expression in various biological research areas.

Key Study Components

Area of Science

Optogenetics
Cell engineering
Gene expression control

Background

Optogenetics allows precise control of cellular functions using light.
Standardization of methods is crucial for reproducibility in research.
The protocol is designed for labs new to optogenetics.
Applications include cancer biology, systems biology, and tissue differentiation.

Purpose of Study

To standardize optogenetic methods for light-induced gene expression.
To provide a comprehensive protocol for researchers.
To enhance the understanding of spatiotemporal control in biological systems.

Methods Used

Selection of genetic components for gene circuit construction.
Assembly of plasmids incorporating light-responsive elements.
Use of genetic engineering and molecular cloning software.
Verification assays to confirm successful gene expression.

Main Results

The protocol enables precise spatial and temporal control of gene expression.
It is adaptable for various engineered mammalian cell systems.
Successful implementation in diverse research applications.
Standardized methods improve reproducibility and reliability in experiments.

Conclusions

The standardized optogenetic protocol is user-friendly and effective.
It broadens the accessibility of optogenetic techniques to more researchers.
This work lays the groundwork for future advancements in optogenetics.

Frequently Asked Questions

What is optogenetics?

Optogenetics is a technique that uses light to control cells within living tissue, typically neurons, that have been genetically modified to express light-sensitive ion channels.

How can this protocol be applied in cancer research?

The protocol allows researchers to manipulate gene expression in cancer cells using light, facilitating studies on tumor behavior and treatment responses.

Is prior experience with optogenetics required?

No, this protocol is designed to be accessible for labs that are new to optogenetics.

What types of genetic components are used in the protocol?

The protocol involves selecting mammalian DNA integration sites, light-responsive elements, and functional genes for circuit construction.

What are the benefits of standardizing optogenetic methods?

Standardization enhances reproducibility, reliability, and the overall understanding of optogenetic applications across various research fields.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

The standardized optogenetic protocol is designed for labs interested in using light as the stimulus to control cellular properties in engineered mammalian cell systems. This protocol is easy to implement in labs otherwise unfamiliar with optogenetics and provides methods for precise spatial temporal control of engineered biological systems. This method has a broad range of application in specific research areas where spatiotemporal information may be important including cancer biology, systems biology, and tissue differentiation.

To begin with, select the genetic components to combine bind into a single gene circuit or plasmid. For example, mammalian DNA integration sites, light responsive elements, or functional genes. After assembling and examining all the necessary features such as start codons, regulatory or translated sequences, use any genetic engineering and molecular cloning software, annotate and store the DNA sequences for later use and as a reference.

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