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ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.

Arnaud Rondelet¹, Andrei Pozniakovsky², Devika Namboodiri¹

¹Max Planck Institute of Molecular Physiology, Dortmund, Germany.

Life Science Alliance

|December 9, 2020

Summary

We developed exogenous/synthetic intronization (ESI) mutagenesis, a new single-step method for efficiently inserting point mutations into bacterial artificial chromosome (BAC) transgenes. This technique simplifies the process, making it faster and more precise for genetic research.

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Bacterial artificial chromosome (BAC)-based transgenes are crucial for studying protein function in eukaryotes.
Current methods for introducing point mutations into BAC transgenes using recombineering are often inefficient and time-consuming.

Purpose of the Study:

To develop a simplified, efficient, and precise method for generating point mutations in BAC transgenes.
To introduce a novel technique called exogenous/synthetic intronization (ESI) mutagenesis.

Main Methods:

Developed ESI mutagenesis, a single-step recombineering method for BAC transgene mutation.
Co-integrates a point mutation of interest with a selectable marker gene within an artificial intron.
Utilizes antibiotic selection to identify successfully mutated BAC transgenes.

Main Results:

ESI mutagenesis enables efficient and precise single-step insertion of point mutations into BAC transgenes.
Cell lines derived from ESI-mutated BACs show equivalent transgene expression to endogenous genes.
The artificial intron is efficiently spliced out in all cells, ensuring functional transgenes.

Conclusions:

ESI mutagenesis offers a robust, high-efficiency, and high-precision method for mutating BAC transgenes.
This technique significantly streamlines the generation of precisely mutated BAC transgenes for research.