Improved Recomibinase Technology for Targeted Marker Free Integration and Founder Line Production for Risk Assessment
Crop Improvement & Utilization Research
2012 Annual Report
1a.Objectives (from AD-416):
The research objectives are to reduce potentially negative effects of transgene insertion and genomic presence. The aim of this proposed research is to investigate the use of novel unidirectional recombinases Bxb1, CinH, ParA and phiC31 to implement a Recombinase-Mediated Cassette Exchange (RMCE) technique for precise integration with simultaneous marker removal. The specific goals are:1) To identify the most efficient pair of recombinase enzymes for dual unidirectional RMCE. .
2)To demonstrate proof of concept with dual unidirectional RMCE in Camelina sativa. .
3)To generate transgenic founder Camelina sativa lines containing the RMCE genetic platform for precise biotech risk assessment.
1b.Approach (from AD-416):
Single copy transgenic Camelina sativa founder lines will be generated containing the selection gene cassette flanked by fused recognition sites. An exchange vector will be biolistcally transformed into the various Camelina sativa founder lines. Recombinase mediated cassette exchange will be examined by negative selection and used to score the most efficient pairs. The most effective Camelina sativa founder lines and recombinase pairs will be published and made publicly available.
The plasmid constructs required for initial camelina transformation were completed. Production of transgenic camelina containing the ‘TAG’ site, required for recombinase mediated targeting, was initiated. Twelve transformants have been identified so far, but all have multiple copies of the ‘TAG’ site. New transformation experiments have been undertaken, with the goal of recovering single copy lines. The pEXCH plasmid, required for Recombinase-Mediated Cassette Exchange with the genomic ‘TAG’ sites, is being constructed. The design will be modified to allow for both biolistic and Agrobacterium-mediated DNA delivery. The compounds required for the conditional negative selection (using marker gene codA) were tested on wild type camelina and the range of concentrations that are not toxic was determined. Finally, the transgenic camelina lines containing the ‘TAG’ sites were grown in the presence of the codA selection agents and as expected, sickened and died. This research relates to Objective 1 of the parent project, the development of recombination systems for plants.