Codon-Optimized FLPo Recombinase for Mammalian Cell Efficiency
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Solution Overview
Problem
The low recombination efficiency of FLP DNA site-specific recombinase in mammalian cells due to its prokaryotic origin, which results in inefficient gene expression and activity, limiting its applications in eukaryotic systems.
Innovation Solution
A codon-optimized FLP (FLPo) gene with enhanced recombinase activity is developed, optimized for mouse codon usage, and engineered to improve translational efficiency, mRNA stability, and reduce CpG content, thereby increasing recombination efficiency in mammalian cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the native FLP gene is used in mammalian cells, then the recombination activity is present, but the recombination efficiency is low due to prokaryotic origin and inefficient gene expression
Solution Approach 1:
The patent applies parameter changes by optimizing the FLP gene's coding sequence for mouse codon usage patterns. This involves changing the nucleotide sequence parameters to match eukaryotic codon preferences, thereby improving translational efficiency and protein expression levels, which directly increases recombination efficiency in mammalian cells
Solution Approach 2:
The patent applies local quality by specifically optimizing certain regions of the FLP gene, particularly the coding sequence, while maintaining other functional regions. The optimization focuses on codon usage, GC content, and regulatory elements specific to mammalian expression, thereby improving local expression efficiency without compromising overall recombination function
2Productivity
If the FLP gene is expressed in mammalian cells, then recombination can occur, but the recombination rate remains low (at most 6% in ES cell clones)
Solution Approach 1:
The patent changes key parameters of the FLP gene including codon optimization for mouse usage, adjustment of GC content, and modification of regulatory sequences. These parameter changes result in significantly improved translation efficiency and protein stability, thereby increasing the recombination rate from less than 6% to substantially higher levels in mammalian cell lines
Solution Approach 2:
The patent creates an optimized copy of the FLP gene (FLPo) that replicates the functional capabilities of the native FLP gene while incorporating improvements in expression efficiency. This optimized copy maintains the essential recombination function while achieving much higher productivity through improved translational efficiency and mRNA stability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The optimized FLP gene demonstrates a significant increase in recombination efficiency, facilitating a wider range of molecular applications, including efficient removal of selection cassettes and multiple gene manipulations in cultured cells and transgenic organisms.
Implementation Method 1
DNA site-specific recombinases (SSRs) are a powerful tool for analyzing gene function in eukaryotes. SSRs recognize specific DNA sequences (recognition sites) and catalyze recombination between two recognition sites.
Implementation Method 2
FLP from Saccharomyces cerevisiae is another SSR that has been used in mammals (Dymecki, Proc. Natl. Acad. Sci. U.S.A. 93 (12):6191-6 (1996)). Similar to Cre, FLP recognizes a distinct 34 bp sequence known as an FRT site, and can mediate the deletion, inversion, and insertion of DNA sequences between two of these sites
Data Source
AI summary
The present invention provides an optimized FLP site-specific recombinase coding sequence and methods for its use. This genetically engineered FLP gene displays a marked increase in recombination efficiency compared to the native FLP gene and is therefore useful in a wide array of molecular applications.