Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Asha  A. Philip; Jin Dai; Sarah P. Katen; John  T. Patton

doi:10.3791/61039

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

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11:40 min

April 17, 2020

DOI: 10.3791/61039-v

Asha A. Philip¹, Jin Dai¹, Sarah P. Katen¹, John T. Patton¹

¹Department of Biology,Indiana University

Overview

This protocol describes a reliable and efficient reverse genetics system for generating recombinant rotaviruses. It details the process for creating recombinant strains that express fluorescent marker proteins, utilizing a minimal number of plasmids.

Key Study Components

Area of Science

Virology
Genetics
Vaccine Development

Background

Rotaviruses are significant pathogens causing gastroenteritis.
Understanding rotavirus replication and pathogenesis is crucial for vaccine development.
Recombinant rotaviruses can serve as valuable tools in research.
Fluorescent reporter proteins can aid in visualizing viral processes.

Purpose of Study

To simplify the generation of recombinant rotaviruses.
To facilitate the study of rotavirus biology.
To develop rotavirus expression vectors for research and vaccine purposes.

Methods Used

Seeding BHKT7 cells onto 12-well plates.
Rinsing cells with PBS and disrupting the monolayer with trypsin-EDTA.
Resuspending cells in GMEM complete medium.
Utilizing a minimal number of plasmids for recombinant virus generation.

Main Results

Successful generation of recombinant rotaviruses expressing fluorescent proteins.
Demonstrated efficiency of the reverse genetics system.
Provided a straightforward method for future rotavirus studies.
Enabled visualization of viral replication processes.

Conclusions

The simplified reverse genetics approach is effective for rotavirus research.
Recombinant rotaviruses can be used to study viral pathogenesis.
This method can contribute to the development of vaccines and expression vectors.

Frequently Asked Questions

What are recombinant rotaviruses?

Recombinant rotaviruses are genetically modified viruses used for research purposes, particularly in studying viral replication and pathogenesis.

How does the reverse genetics system work?

The reverse genetics system allows researchers to create recombinant viruses from plasmid DNA, facilitating the study of viral functions and interactions.

What is the significance of fluorescent reporter proteins?

Fluorescent reporter proteins enable visualization of viral processes in live cells, aiding in the understanding of rotavirus biology.

What cell line is used in this protocol?

The protocol utilizes BHKT7 cells for the generation of recombinant rotaviruses.

Can this method be used for other viruses?

While this method is specifically designed for rotaviruses, similar reverse genetics approaches can be adapted for other viral systems.

What are the applications of recombinant rotaviruses?

Recombinant rotaviruses can be used in vaccine development, studying viral pathogenesis, and as expression vectors for research.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

This protocol describes a reliable and efficient reverse genetics system for making recombinant rotaviruses. It also explains how to make recombinant rotaviruses that express fluorescent marker proteins. This method is simple requiring a minimal number of plasmids and can generate recombinant rotaviruses that express separate fluorine proteins as well as also viral proteins.

Begin by seeding BHKT7 cells onto 12-well plates. Rinse a freshly confluent monolayer of cells with PBS. Then disrupt the monolayer with trypsin-EDTA solution and resuspend the cells in five milliliters of GMEM complete medium.

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