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Recombinatorial cloning using heterologous lox sites.
Robert W Siegel1, Nileena Velappan, Peter Pavlik
1Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Genome Research
|June 3, 2004
Summary
This study introduces a novel Cre/loxP recombination system for efficiently cloning affinity reagents. The system ensures high-fidelity gene transfer with minimal impact on reagent functionality, facilitating proteome characterization.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomics
Background:
- Traditional cloning methods present bottlenecks for high-throughput gene transfer.
- Existing recombination systems are not optimized for cloning affinity reagents from display libraries.
- Affinity reagents are crucial for proteome functional characterization.
Purpose of the Study:
- To develop a Cre/loxP-based recombination system for efficient cloning of affinity reagents.
- To ensure minimal impact of recombination signals on affinity reagent expression and function.
- To facilitate high-throughput downstream applications of display-selected affinity reagents.
Main Methods:
- Utilized a Cre/loxP-based system with heterologous loxP sites (Lox 2372 and Lox WT) flanking an affinity reagent (scFv).
- Incorporated a conditionally lethal gene (SacB) to enable efficient transfer to destination vectors.
- Validated minimal impact of translated lox sites on scFv expression and functionality.
Main Results:
- Developed a high-fidelity gene transfer system for affinity reagents.
- Demonstrated that translated lox sites have minimal impact on scFv functionality.
- Achieved efficient transfer of affinity reagents to destination vectors.
Conclusions:
- The novel Cre/loxP system facilitates high-throughput cloning of affinity reagents selected from display libraries.
- This approach is applicable to general recombinatorial cloning for genomic purposes.
- The system enhances the utility of affinity reagents in proteome research.