Improved Recomibinase Technology for Targeted Marker Free Integration and Founder Line Production for Risk Assessment
Crop Improvement & Utilization Research
Project Number: 5325-21000-020-01
Start Date: Sep 01, 2010
End Date: Aug 31, 2014
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.
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 biolistically 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.