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Development of a Single Vector Containing cre Recombinase and Two Functional lox Sites: Active cre Produced in Eukaryotic but not Prokaryotic Systems,
Stan Kaczmarczyk, Jeffrey E. Green (NCI),DHHS Reference No. E-172-00/0 filed 04 Apr 2001
The bacterial recombinase cre will recombine lox sites within bacteria. Since this will occur with extremely low levels of cre, it has not been possible to place the cre gene within a vector which also contains two lox recombination site and clone the construct in bacteria. Therefore, in order to use cre-lox technology, the use of two separate vectors has been required—one containing cre and another containing the lox sites. The inventors have devised a strategy to generate vectors that contain cre in a form which is not translated in bacteria thereby allowing for the co-existence of two lox sites within the same vector. Under these circumstances, the vector can be cloned and grown in bacteria enabling experiments to be conducted in eukaryotic cells using just one vector instead of two separate vectors. This system provides a significant advantage in performing many types of experiments.
In in vitro transfection experiments, only one vector needs to be incorporated into a cell instead of two separate vectors. This overcomes the inherent problem of trying to transfect two vectors into one cell, where the relative ratios of the two vectors which enter the cells can vary widely. Of even greater significance is the application of this technology to transgenic animal work where the incorporation of one vector into a line of transgenic animals is all that is required, instead of the generation of two separate lines of transgenic animals which then must be crossed to produce an animal which contains both constructs. In addition, this technology can be applied to gene therapy approaches in which the tissue-specific expression of a therapeutic gene can be activated by cre contained within the same construct. The technology allows generation of one vector which contains the cre-variant and two lox sites enabling one to either switch the expression of one gene to another gene and/or amplify the expression of a particular gene to high levels in a tissue specific manner.
This research has appeared, in part, in Kaczmarczyk and Green, Nucleic Acids Res 2001 Jun 15;29(12):E56.Dated: September 28, 2001. Jack Spiegel, Director, Division of Technology Development and Transfer,Office of Technology Transfer,National Institutes of Health.