In order to speed up the process of developing the crops with multiple transgenic traits, Dr. David W. Ow’s group from the South China Botanical Garden (SCBG) under the Chinese Academy of Sciences has established a recombinase-mediated gene stacking system.

In this system, the Cre recombinase is required to excise DNA no longer needed (such as selectable markers and plasmid backbones) after each round of site-specific integration.

The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. Cre-mediated deletion of lox-site flanked DNA is generally efficient, but depends on the cre-expressing donor line used in the cross.

In former study, a cre line was found less efficient than expected, resulting in all F1 plants chimeric with a mixture of cells with or without cre-lox recombination.

This is probably because the cre line has become less effective over time. Although gene silencing over the generations could have occurred with any transgene, there is some reason to suspect that high expression of the DNA-binding recombinase, Cre, could interfere with normal chromosome activities.

To overcome the problem occurred in cre lines, Dr. David W. Ow and the former Ph.D. student Maryam Rajaee considered a split-cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization.

As some Cre fragment complementation approaches had been patented, to insure that the recombinase-mediated gene stacking system retains its freedom to operate, they tested for new locations to split Cre into complementing fragments.

With several attempts, Dr. Ow and his former student finally found a new location, between Lys244 and Asn245, for splitting Cre. Furthermore, they proved that the new split-Cre pair could reconstitute activity that was comparable to that of wild-type Cre control.

Obviously, this new approach will be very useful in excising unneeded DNA with a high efficiency and moreover, can add feasibility and flexibility to the ‘open-source’ gene stacking system that is free to operate for commercial crop improvement.