En Passant Mutagenesis for Markerless DNA Design in Escherichia coli
- Detailed Technology Description
- Bacterial artificial chromosomes (BACs) have made it possible to rapidly and efficiently introduce mutations to large DNA sequences.
- Others
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None
- *Abstract
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Bacterial artificial chromosomes (BACs) have made it possible to rapidly and efficiently introduce mutations to large DNA sequences. A major disadvantage of most methods using BAC mutagenesis is the retention of selection markers. To avoid marker retention, long, foreign sequences, FRT or loxP sites have been employed to excise positive selection markers with recombinases. However, one copy of the FRT or loxP site remains, limiting the use of these techniques for later modifications as they can interrupt parental sequences. Thus, a more efficient method that allows for markerless mutagenesis on large sequences would be advantageous.
Researchers at Cornell have developed two modes for markerless DNA manipulation:
- Researchers have inserted parental viral sequences between the bacterial origin of replication and a gene essential for the maintenance of the BAC that results in duplication of sequences originally up and downstream of the mini-F insertion within mini-F sequences. Recombination events between these homologous sequences would separate the origin of replication and the positive selection marker, usually the antibiotic resistance gene. After transfection into eukaryotic cells and virus reconstitution, two successful recombination events result in restoration of the parental sequence without undesired vector sequences. The new method allows for the use of mini-F based replicons as tools for genetic manipulation without unwanted affects. (D-3715)
- Alternatively researchers have found a way to do highly efficient PCR-based, DNA engineering without the retention of unwanted sequences by combining Red recombination and cleavage with the homing endonuclease I-SceI. The positive selection markers used to introduce the desired target modification are removed by I-SceI cleavage and intramolecular Red recombination. (D-3688).
Potential Applications & Advantages
Markerless DNA manipulation can be used for:
- Generation of transgenic animals;
- Gene therapy;
- Vaccine vectors.
- *Licensing
- Phillip Owhpo62@cornell.edu607-254-4508
- Country/Region
- USA
