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Reproductive Cloning01:27

Reproductive Cloning

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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 4, 2026

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics
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FX cloning: a simple and robust high-throughput cloning method for protein expression.

Eric R Geertsma1

  • 1Institute of Biochemistry, Biocenter N220, Goethe-University Frankfurt, Frankfurt/M., Germany.

Methods in Molecular Biology (Clifton, N.J.)
|January 8, 2014
PubMed
Summary

FX cloning is an efficient method for cloning open reading frames (ORFs) to express proteins. This technique streamlines high-throughput projects and single targets, adding minimal amino acids for versatile expression vectors.

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Area of Science:

  • Molecular Biology
  • Protein Expression
  • Biotechnology

Background:

  • The increasing volume of gene sequence data necessitates efficient protein screening.
  • High-throughput cloning tools are crucial for identifying novel proteins.
  • Existing cloning methods often have limitations in versatility and efficiency.

Purpose of the Study:

  • To introduce FX cloning, a novel method for open reading frame (ORF) cloning.
  • To develop a versatile and efficient tool for protein expression vector construction.
  • To overcome limitations of existing cloning techniques in high-throughput settings.

Main Methods:

  • FX cloning utilizes type IIS restriction enzymes and negative selection markers.
  • The procedure is a one-pot reaction, completed in under 3 hours.
  • It accepts both sequence-verified ORFs and PCR products for subcloning.

Main Results:

  • FX cloning minimizes the addition of amino acids to the N- and C-termini of target proteins (one amino acid per side).
  • This method enables the generation of both N- and C-terminal translational fusions using a single primer pair or PCR product.
  • The procedure is highly efficient, economical, and robust, suitable for various expression systems.

Conclusions:

  • FX cloning offers a streamlined, efficient, and versatile approach for protein expression.
  • The method is well-suited for both high-throughput and routine cloning applications.
  • FX cloning simplifies vector construction while maintaining protein integrity.