Improved Recombinase Technology for Targeted Marker Free Integration and Founder Line Production for Risk Assessment
Crop Improvement & Utilization 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 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 goal of this research, which contributes to objective 2A of the in-house project, is to establish a recombinase platform in soybean 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 experience in soybean transformation that is critical to project success, and.
2)construction of the novel transformation vectors needed for the recombinase platform.