Precision of Transgene Integration by Site-Specific Recombination !
Possibility of site-specific recombination and homologous recombination process has been exploited in recent days to potentially increase transgene integration. It has been developed into the genetic engineering tool for higher eukaryotes.
Despite the efficiency of using Agrobacterium for transformation, integration is still random and has no control over the impact of genomic position on transgene silencing. Recently, the combination of homologous and site-specific recombination has emerged as a powerful chromosome-engineering technology in mammals and insects.
By exploiting homologous recombination strategy, foreign DNA can be integrated into predetermined chromosomal locations by homologous recombination, and site-specific recombination is used for subsequent modifications of transgenic loci. In this technology, feasibility of cre/Iox P and FLP/FRT systems have been widely exploited in wide range of organisms.
Although the combination of homologous and site-specific methods are yet to be established in plants, but still both FLP are used to integrate new transgene into pre-existing transgene loci in the genome. It can be demonstrated that precise transgene placement is feasible. This is absolutely required for precision and predictability in the production of transgenic crops.
Site-specific recombination is a natural process existing commonly in prokaryotes and lower eukaryotes. Natural functions of such systems include integration of a bacteriophage into the host genome, maintenance of copy number etc. Similarly, FLP/FRT system related to the acceleration of replication process in yeast system is well known.
Both FLP/FRT and cre/IoxP site-specific recombination systems have been widely used in plants to enhance precise transgene integration. Initial studies on site-specific recombination system in plants used both cre/IoxP from bacteriophage and FLP/FRT from yeast two component systems in order to demostrate functions of ere in tobacco, FLP in maize and rice. These two systems have been used successfully for the removal of selectable markers from nuclear chloroplast and DNA in transgenic plants.
Site-specific recombination strategy has been successfully implicated by using cre/IoxP to integrate T-DNA into predetermined chromosomal loci in Arabidopsis. Two approaches were taken for formation of substrate T-DNA within plant cells. One was border recombination in which a single IoxP site was placed in the T-DNA and other was intramolecular recombination within the T-DNA containing two IoxP sites.
Due to insufficient transformation, the expression cassette containing IoxP-35S-ATG-IoxP was manipulated further by amplifying single stranded T-DNA into the double stranded for successful site specific integration. Once T-DNA is amplified, the majority of the recovered T-DNA molecules from maize are the product of site-specific recombination.
In the extended new strategies, two IoxP sites, each mutated in left side in one and right side in other were synthesized. This method was found to be successful in tobacoo and also in embryonic stem cells. Another refined strategy that is very effective at increasing integration at a single-site locus depends on inactivation of recombinase expression as a result of integration.