Improved Recombinase 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 Glycine max. .
3)To generate transgenic founder Glycine max lines containing the RMCE genetic platform for precise biotech risk assessment.
1b.Approach (from AD-416):
Single copy transgenic Glycine max 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 Glycine max founder lines. Recombinase mediated cassette exchange will be examined by negative selection and used to score the most efficient pairs. The most effective Glycine max founder lines and recombinase pairs will be published and made publicly available.
The plasmid constructs required for initial soybean transformation were completed. Production of transgenic soybean containing the ‘TAG’ site required for recombinase mediated targeting has been initiated. Two transgenic lines carrying the required ‘TAG’ site have been identified so far. One of the lines has a single copy of the ‘TAG’ site and it is being grown for seed increase. Transformation experiments continue to obtain more ‘TAG’ lines with single copy transgenes. The pEXCH plasmid, required for Recombinase-Mediated Cassette Exchange with the genomic ‘TAG’ sites, is being constructed. The design will be modified to allow both biolistic and Agrobacterium-mediated DNA delivery. The compounds required for the conditional negative selection (using marker gene codA) were tested on wild type soybean and the range of concentrations that are not toxic was established. This research relates to Objective 1 of the parent project, the development of recombination systems for plants.