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Nucleic Acids Research, 2000, Vol. 28, No. 7 E19-e19
© 2000 Oxford University Press

A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

Sangdun Choi*, Dilara Begum1, Heather Koshinsky2, David W. Ow2 and Rod A. Wing1

Division of Biology 147-75, California Institute of Technology, Pasadena, CA 91125, USA, 1Departments of Agronomy and Biological Sciences, Clemson University, Clemson, SC 29634, USA and 2Plant Gene Expression Center, USDA/ARS-UC Berkeley, 800 Buchanan Street, Albany, CA 94710, USA

With current plant transformation methods (Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incor­porating the Cre/lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted ‘position effects’ caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.

* To whom correspondence should be addressed. Tel: +1 626 395 4504; Fax: +1 626 796 7066; Email: schoi@its.caltech.edu


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