1a. Objectives (from AD-416)
The long term goal of our research is to develop an efficient, stable and deployable Tissue-Specific Transgene Removal and Containment System (TRECS) to comprehensively address the concerns of transgene flow in crops that can be vegetatively reproduced. This goal can be best achieved by understanding the gene regulatory mechanisms underlying the TRECS process and minimizing unfavorable interactions among cis-regulatory elements within the TRECS system. Specifically, we will take advantage of two enhancer-blocking insulators we identified, as well as sequence-diverged but functionally identical petunia PtAGIP promoter we characterized to comprehensively address the problems that hamper the TRECS performance with two objectives: 1) establish a functional and operable TRECS system in plants through the use of enhancer-blocking insulators and the PtAGIP promoter and 2) further improve the established TRECS efficiency through characterization and optimization of molecular scissors efficiency.
1b. Approach (from AD-416)
Tobacco NtAGIP will be first replaced with petunia PtAGIP promoter that shares the identical tissue specificity and activity but has only 50% sequence identity with NtAGIP. This replacement should minimize a gene silencing-related problem due to its diverged DNA sequence. Genetic insulators, TBS and EXOB, will be inserted into the different sites in the Tissue-Specific Transgene Removal and Containment System (TRECS) constructs to flank the PtAGIP::iCre transcriptional unit and tested for blocking promoter-promoter crosstalk within TRECS. This work aims at establishing an operable TRECS platform which can be used as a platform for further improvement of TRECS efficiency. Finally, co-expression of multiple scissors genes through the use of a bi-directional promoter in TRECS (e.g. Flp with Cre) and fusion of specific nuclear localization signals to scissors proteins will be analyzed.
3. Progress Report
The objective of the project is to focus on the optimization of the Tissue-Specific Transgene Removal and Containment System (TRECS) gene excision efficiency. We have inserted two enhancer-blocking insulators, DNA sequences previously identified in our lab, into the existing TRECS vectors to generate new versions, which have been introduced into tobacco. Over 150 transgenic plants have been generated, rooted, and transferred to the greenhouse. The detailed examination of the gene excision efficiency is in progress. The ADODR has monitored activities through meetings.