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In-vitro Mutagenesis01:16

In-vitro Mutagenesis

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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.
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Related Experiment Video

Updated: Oct 29, 2025

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
14:06

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Published on: November 12, 2012

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Gene Switching and Essentiality Testing.

Amanda Claire Brown1,2

  • 1Texas A&M Veterinary Medical Diagnostic Laboratory (TVDML), College Station, TX, USA. amanda.brown@tvmdl.tamu.edu.

Methods in Molecular Biology (Clifton, N.J.)
|July 8, 2021
PubMed
Summary

Identifying essential genes in mycobacteria is crucial but challenging. This study presents a gene switching method to confirm essentiality by introducing a second copy of the gene, allowing deletion of the original and confirming essentiality if no transformants arise.

Keywords:
Essentiality testingGene homologsGene switching

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Identifying essential genes in mycobacteria is vital for research.
  • Confirming gene essentiality is difficult due to lethal phenotypes upon deletion.

Purpose of the Study:

  • To describe protocols for confirming gene essentiality in mycobacteria using gene switching.
  • To validate gene functionality and identify essential operon members.

Main Methods:

  • Construction of a strain with the sole functional gene copy on an integrated plasmid (Δ'int).
  • Introduction of a second gene copy via an integrating vector to enable chromosomal gene deletion.
  • Application of the gene switching method to replace the integrated vector.

Main Results:

  • Absence of transformants after gene switching confirms essentiality of the targeted gene.
  • The method successfully confirms gene essentiality.

Conclusions:

  • The described gene switching protocol provides a reliable method for confirming essential gene identification in mycobacteria.
  • This technique is also applicable for validating gene homologs and identifying essential genes within operons.