Improved Recombinase Technology for Targeted Marker Free Integration and Founder Line Production for Risk Assessment
Crop Improvement and Genetics Research
2011 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.
Camelina sativa is a mustard plant that produces oil-rich seeds that show promise as sources of biofuel. The goal of this research, which contributes to objective 2A of the in-house project, is to establish a recombinase platform in camelina that will allow.
1)the removal of marker genes and other unneeded foreign DNA, and.
2)the insertion of new genes into chromosomal regions that support their activity and inheritance. The project has been initiated with new funding in FY11 by beginning.
1)recruitment of a postdoctoral associate with the experience in camelina/Arabidopsis transformation and recombinase experience that is critical to project success, and .
2)construction of the novel transformation vectors that contain the recombinase platform.