A multi-step strategy for BAC recombineering of large DNA fragments.

Recombineering techniques have been developed to modify bacterial artificial chromosomes (BACs) via bacterial homologous recombination systems, simplifying the molecular manipulations of large DNA constructs. However, precise modifications of a DNA fragment larger than 2-3 kb by recombineering remain a difficult task, due to technical limitations in PCR amplification and purification of large DNA fragments. Here, we describe a new recombineering strategy for the replacement of large DNA fragments using the commonly utilized phage/Red recombination host system. This approach involved the introduction of rare restriction enzyme sites and positive selection markers into the ends of a large DNA fragment, followed by its release from the donor BAC construct and integration into an acceptor BAC. We have successfully employed this method to precisely swap a number of large DNA fragments ranging from 6 to 40 kb between two BAC constructs. Our results demonstrated that this new strategy was highly effective in the manipulations of large genomic DNA fragments and therefore should advance the conventional BAC recombineering technology to the next level.

[1]  R. Moriggl,et al.  A detailed protocol for bacterial artificial chromosome recombineering to study essential genes in stem cells. , 2008, Methods in molecular biology.

[2]  Shailaja N Hegde,et al.  Co-targeting a selectable marker to the Escherichia coli chromosome improves the recovery rate for mutations induced in BAC clones by homologous recombination. , 2004, BioTechniques.

[3]  Nancy A. Jenkins,et al.  Simple and highly efficient BAC recombineering using galK selection , 2005, Nucleic acids research.

[4]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Jiyue Zhu,et al.  Transcriptional silencing of a novel hTERT reporter locus during in vitro differentiation of mouse embryonic stem cells. , 2006, Molecular biology of the cell.

[6]  N. Heintz,et al.  Bac to the future: The use of bac transgenic mice for neuroscience research , 2001, Nature Reviews Neuroscience.

[7]  A. Stewart,et al.  Rapid modification of bacterial artificial chromosomes by ET-recombination. , 1999, Nucleic acids research.

[8]  D. Court,et al.  A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. , 2001, Genomics.

[9]  I. Huhtaniemi,et al.  Assisted large fragment insertion by Red/ET-recombination (ALFIRE)—an alternative and enhanced method for large fragment recombineering , 2007, Nucleic acids research.

[10]  A. L. La Spada,et al.  Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis. , 2006, Gene.

[11]  Nancy A. Jenkins,et al.  Recombineering: a powerful new tool for mouse functional genomics , 2001, Nature Reviews Genetics.

[12]  V. Beneš,et al.  Point mutation of bacterial artificial chromosomes by ET recombination , 2000, EMBO reports.

[13]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[14]  M. Leiby,et al.  Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome , 2009, Nucleic acids research.

[15]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[16]  M. Leiby,et al.  A New Positive/Negative Selection Scheme for Precise BAC Recombineering , 2009, Molecular biotechnology.

[17]  Giuseppe Testa,et al.  DNA cloning by homologous recombination in Escherichia coli , 2000, Nature Biotechnology.