Current members of the Parrott lab pose outside their laboratory. The paper’s senior author, Dr. Monica Schmidt, is currently at the Danforth Center in St. Louis.
A Comparison of Strategies for Transformation with Multiple Genes via Microprojectile-Mediated Bombardment
As plant genetic transformation advances in scope, there will be a greater need to engineer with multiple genes, either for trait-stacking or for the engineering of metabolic pathways requiring multiple genes. There has been tremendous progress in designing vectors that permit the easy assembly of multiple genes into one cassette, particularly through the use of multiple cloning areas consisting of sites for intron- and intein-encoded endonucleases, also known as meganucleases, along with an associated set of shuttle vectors. These have been used to a limited extent with both Agrobacterium- and microprojectile-mediated bombardment. However, very little is known about the relative efficiency of different strategies for the simultaneous engineering of multiple genes. We have previously engineered BAC DNA in excess of 100 kb long via the gene gun, and these have integrated intact into a single locus. Our current objective was to determine if a multiple-gene-cassette would behave likewise, or if engineering with separate cassettes would be more effective for using the gene gun. Multiple gene cassettes do not appear to be stable as currently constructed, a shortcoming that might be reduced by minimizing DNA repeats within the cassette. In the near term, shooting the multiple cassettes independently may be the better option.
Monica A. Schmidt, Peter R. LaFayette, Barbara. Artelt, and Wayne A. Parrott. A Comparison of Strategies for Transformation with Multiple Genes via Microprojectile-Mediated Bombardment, In Vitro Cellular & Developmental Biology-Animal 44:162-168, 2008.
