Optimizing integration events in the Cre/loxP system

Current methods of transgenesis revolve around microinjection and homologous recombination procedures. These commonly used techniques suffer from a lack of precision or wide range application, respectively. An alternative method for transgenesis is the use of the Cre/loxP system. This site specific binary recombinase system that has shown a wide range of application due to the recombination reaction requiring only the Cre enzyme and the loxP site, a unique 34bp DNA binding domain. Cre can be thought of as an enzyme that can recognize and bring together two loxP sites. The two sites are then cut in half and opposite parts exchanged and bound back together. An advantage of this enzymatic system is that it is capable of conservative site-specific excision and integration events. This is a conservative reaction, meaning that no DNA is added or lost during the exchange, unlike results seen for microinjection or homologous recombination. Unfortunately, in the wild-type form the excision reaction is favored. Through the modification of the loxP site, the Cre system has been used to bring two separate loxP sites together for permanent DNA insertions. The objective was to modify the loxP sites enough so that when the two are cut and exchanged a new unique loxP site is generated that will no longer be recognized by the Cre. This inhibits the excision reaction thereby trapping the inserted DNA. This study has created an in vivo system that eliminates both the positional effect and allowed screening of mutant loxP sites that favor integration. Fifty different mutant loxP sites combinations have been tested using the wild type as the control. To date the most efficient combinations are lox510:17 and lox15:17, showing 1421 and 1523 times more effective relative rates of integration when normalized against wild type. By combining this information with the common techniques of transgenesis, it may be possible to give microinjection the precision of homologous recombination without its limitations.