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Updated: Dec 13, 2025

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

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

Published on: January 8, 2015

Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs.

Remya R Nair¹, Charlotte Tibbit¹, David Thompson¹

¹Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire OX11 0RD, UK.

Methods (San Diego, Calif.)

|July 30, 2020

Summary

This summary is machine-generated.

Researchers developed methods to stabilize large microsatellite repeat expansions in bacterial vectors, crucial for modeling neurological disorders like C9orf72-ALS. This facilitates cloning for genetic engineering and disease research.

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Last Updated: Dec 13, 2025

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Published on: January 8, 2015

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Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

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Published on: July 12, 2012

Area of Science:

Genetics
Molecular Biology
Neuroscience

Background:

Microsatellite repeat expansions cause heritable neurological disorders.
Instability of these repeats in bacterial vectors hinders disease modeling and genome engineering.

Purpose of the Study:

To develop efficient methods for cloning and maintaining large microsatellite repeat expansions.
To improve the stability of C9orf72 gene hexanucleotide repeats in bacterial constructs.

Main Methods:

Utilized a BAC construct from a C9orf72-ALS patient.
Optimized bacterial growth conditions for repeat retention.
Investigated sub-cloning into linear vectors and replication orientation effects.

Main Results:

Established efficient repeat sizing and bacterial growth protocols.
Demonstrated improved repeat stability via sub-cloning into linear vectors.
Confirmed stability is dependent on DNA replication orientation relative to the repeat.

Conclusions:

The presented methods offer a straightforward approach to maintaining large microsatellite repeat expansions.
These findings aid in the efficient cloning of unstable repeats for disease research and genetic engineering.