S'identifier

Recombineering and Gene Targeting

Vue d'ensemble

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based on stretches of similar or identical sequences. Together with gene targeting, which takes advantage of endogenous HR to alter an organism’s genome at a specific loci, HR-based cloning techniques have greatly improved the speed and efficacy of high-throughput genetic engineering.

In this video, we introduce the principles of HR, as well as the basic components required to perform a recombineering experiment, including recombination-competent organisms and genomic libraries such as bacterial artificial chromosomes (BAC). We then walk through a protocol that uses recombineering to generate a gene-targeting vector that can ultimately be transfected into embryonic stem cells to generate a transgenic animal. Finally, several applications that highlight the utility and variety of recombineering techniques will be presented.

Procédure

Cloning by homologous recombination, or recombineering, has greatly improved researchers’ ability to conduct high-throughput genetic engineering. Classical molecular cloning requires digestion of vectors and inserts with the same restriction enzymes to generate compatible ends for “recombination,” but especially when trying to isolate a region from a longer sequence, such as a stretch of genomic DNA, there are not always restriction enzymes that will cut uniquely around the region of interest, and not wit

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Tags
RecombineeringGene TargetingCloningHomologous RecombinationGenetic EngineeringMolecular CloningRestriction EnzymesGenetic RecombinationRecombination ProtocolGene targeting VectorApplications Of RecombineeringHomologous ChromosomesGenetic Diversity

Passer à...

0:00

Overview

1:05

Principles of Homologous Recombination

3:20

Recombineering a Gene Targeting Vector

5:54

Applications

8:28

Summary

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