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Reproductive Cloning

Reproductive Cloning

Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT).
Somatic Cell Nuclear Transfer
In SCNT, an egg cell is taken from an animal and its nucleus is removed, creating an enucleated egg. Then a somatic...

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Related Experiment Video

Updated: May 5, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method.

Xiaoxue Li¹, Yutian Peng², Wen Xue³

¹Institute of Biochemistry and Molecular Biology, Hengyang Medical School, University of South China; Clinical Research Institute, The Affiliated Nanhua Hospital, Hengyang Medical School, University of South China.

Journal of Visualized Experiments : Jove

|June 30, 2025

Summary

This summary is machine-generated.

CRISPRshuttle cloning is a new method for transferring DNA fragments between plasmids without PCR amplification. This technique significantly speeds up the creation of genome-wide plasmid libraries for gene function studies.

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Last Updated: May 5, 2026

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Published on: January 8, 2015

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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

Area of Science:

Molecular Biology
Genomics
Synthetic Biology

Background:

Genome-wide plasmid libraries are essential for systematic gene functional characterization.
Current methods for DNA fragment transfer are time-consuming and technically demanding due to PCR amplification requirements.

Purpose of the Study:

To develop a novel, high-throughput cloning method for efficient genome-wide plasmid library generation.
To streamline the process of inter-vector DNA fragment transfer without PCR amplification.

Main Methods:

Developed CRISPRshuttle cloning, a method utilizing a CRISPRshuttle cassette for DNA fragment transfer.
Employed Cas9-mediated cleavage to excise target DNA fragments from donor plasmids.
Utilized Gibson assembly for inserting excised fragments into CRISPRshuttle-compatible vectors.

Main Results:

CRISPRshuttle cloning demonstrated over 94% efficiency.
Two researchers generated approximately 300 plasmids in 7 days using this method.
The protocol involves two sequential test tube reactions before bacterial transformation.

Conclusions:

CRISPRshuttle cloning offers an efficient, adaptable, and cost-effective solution for DNA fragment transfer.
This method significantly accelerates the generation of genome-wide plasmid libraries.
Facilitates high-throughput functional genomics research by simplifying library construction.