2007 Annual Report 1a.Objectives (from AD-416) Identify highly effective enhancer insulators to prevent interaction among promoters in the Transgene Removal and Containment System (TRECS), and bacterial transcriptional repression systems to repress basal level expression of Cre and Flp in E. coli and Agrobacterium. Incorporate identified enhancer insulators and transcriptional repression systems into existing TRECS to improve its performance and utilization. 1b.Approach (from AD-416) Several potential nuclear matrix attachment regions (MARs) and putative enhancer insulators (EI) characterized in plants factors will be cloned and inserted between the 35S and AGIP promoters in 35S/AGIP::GUS construct, where the adjacent 35S promoter activates the flower-specific AGIP::GUS in vegetative tissue. Their ability to insulate the AGIP promoter from interference by the 35S promoter will be evaluated through analyzing GUS expression in leaf, root, and flower tissues through both histochemical staining and RT-PCR. Those that are able to prevent the AGIP::GUS vegetative expression will be identified as candidate EIs. A tetracycline repressor (tetR) that represses tetA expression by binding tet operators (tetO) in the tetA promoter will be first analyzed. Three copies of the tetO will be added to the 3’ end of the AGIP to create an AGIP-tetO promoter that is then placed in front of Cre or Flp. The entire AGIP-tetO::Cre and AGIP-tetO::Flp fragments will be respectively inserted into A-TRECS-tetR vector, where the tetR (constitutively expressed in bacteria) is inserted to the outside of a T-DNA border in the TRECS vector. TetR molecules are expected to bind tetO in the AGIP-tetO promoter and completely repress the basal level expression of Cre or Flp, stabilizing TRECS constructs in bacteria. The identified EIs and repression systems will be incorporated into the existing TRECS to build a new generation of the TRECS platform which will be used for analyzing and comparing excision efficiency among different scissors genes, optimizing and engineering the identified scissors’ efficiency. 3.Progress Report This report serves to document research conducted under a grant funded by the USDA CSREES BRAG program. Additional details of research can be found in the report for the parent project 1931-21000-011-00D, Genetic Improvement of Fruit Crops. Progress was monitored by the ADODR for this in-house research through full participation. Gene flow into wild relative species from transgenic crops is considered as a potential risk for environmental and ecological safety. This research primarily focuses on the development of tissue-specific transgene removal and containment system (TRECS). The major factor that influences TRECS efficiency is promoter-promoter interference with TRECS transgene, and mitigating this problem is one of the high research priorities proposed. We have identified, isolated, and incorporated four potential chromatin/enhancer insulators into binary vectors to create at least 20 constructs. These constructs have been introduced into plants, and their function in preventing promoter-promoter interference in TRECS constructs is currently under investigation. In addition, we have built a new binary vector to address another proposed research in the proposal. Progress of the project was monitored through a lab meeting.