Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

In-vitro Mutagenesis01:16

In-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.
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Gene conversion is a key driver of diversity hotspots in <i>M. tuberculosis</i> antigens and virulence-associated loci.

bioRxiv : the preprint server for biology·2026
Same author

Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection.

Proceedings of the National Academy of Sciences of the United States of America·2025
Same author

Mycobacterium smegmatis NucS-promoted DNA mismatch repair involves limited resection by a 5'-3' exonuclease and is independent of homologous recombination and NHEJ.

Nucleic acids research·2024
Same author

Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection.

bioRxiv : the preprint server for biology·2024
Same author

Phase variation as a major mechanism of adaptation in <i>Mycobacterium tuberculosis</i> complex.

Proceedings of the National Academy of Sciences of the United States of America·2023
Same author

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Proceedings of the National Academy of Sciences of the United States of America·2022

Related Experiment Video

Updated: May 30, 2026

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Targeted chromosomal gene knockout using PCR fragments.

Kenan C Murphy1

  • 1Department of Microbial and Physiological systems, University of Massachusetts Medical School, Worcester, MA, USA. kenan.murphy@umassmed.edu

Methods in Molecular Biology (Clifton, N.J.)
|August 5, 2011
PubMed
Summary

Recombineering enables precise genetic modifications in Escherichia coli, including efficient gene knockouts using PCR fragments and the phage lambda Red recombination system. This technology, combined with Cre and Flp systems, allows for versatile marked and precise gene deletions in microbial chromosomes.

More Related Videos

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
08:25

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Published on: April 8, 2017

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
09:52

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Published on: July 12, 2013

Related Experiment Videos

Last Updated: May 30, 2026

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
08:25

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Published on: April 8, 2017

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
09:52

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Published on: July 12, 2013

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetic Engineering

Background:

  • Recombineering technology allows for extensive genetic modifications in Escherichia coli.
  • Chromosomal gene knockouts are a fundamental genetic modification.
  • Existing methods require efficient and precise gene deletion strategies.

Purpose of the Study:

  • To describe the protocols for performing gene knockouts in Escherichia coli using recombineering.
  • To detail the use of the phage lambda Red recombination system for gene deletion.
  • To explain the integration of site-specific recombination systems for enhanced precision.

Main Methods:

  • Utilizing PCR fragments with selectable drug markers flanked by homologous DNA sequences.
  • Expressing the phage lambda Red recombination system in vivo from a plasmid.
  • Employing Cre and Flp site-specific recombination systems for marked and precise knockouts.

Main Results:

  • High efficiency of gene deletion using the phage lambda Red system.
  • Successful construction of marked and precise gene knockouts.
  • Development of protocols for PCR substrate design, strain generation, and clone verification.

Conclusions:

  • Recombineering is a powerful and versatile technology for Escherichia coli genetic modification.
  • The described protocols facilitate efficient and precise chromosomal gene knockouts.
  • This approach enables advanced genetic engineering in microbial systems.