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United States Department of Agriculture

Agricultural Research Service

Title: A New Approach for the Identification and Cloning of Genes: the P Bac Wich System Using Cre/lox Site-Specific Recombination

Authors
item Choi, Sangdun - CIT, PASADENA CA
item Begum, Dilara - CLEMSON UNIV SC
item Koshinsky, Heather - USDA PGEC
item Ow, David
item Wing, Rod - CLEMSON UNIV SC

Submitted to: Nucleic Acids Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 10, 1999
Publication Date: April 1, 2000

Interpretive Summary: This work tested the feasibility of transferring large DNA segments into the eukaryotic genome through site-specific integration. A bacterial artificial chromosome (BAC) vector was modified for site-specific integration into eukaryotic genomes. Large segments of DNA were cloned into the BAC vector the resulting clones were transformed fungal and plant genomes. Site-specific integration into fungal and plant genomic target sites was shown.

Technical Abstract: 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 incor porating 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.

Last Modified: 10/21/2014
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