We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can over-produce bioactive cytokine after DNA delivery in mice and macaques. We explored the biology of IL-15 and showed that efficient production of IL-15 is possible only by co-expression in the same cell with the IL-15 Receptor-alpha. We also showed that a second form of IL-15 (SSP IL-15) previously identified in humans and rodents as intracellular or nuclear IL-15 is also efficiently secreted from the cells when co-expressed with the IL-15 Receptor alpha. These results shed new light in the biology and regulation of IL-15 and provide methods for the efficient production and clinical application of this cytokine. We have used optimized expression vectors to express IL-12 cytokine in animals. Efficient expression results in bioactive levels, which increase immune response after DNA vaccination, thus becoming important molecular adjuvant for our vaccines. We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have identified the cellular factor responsible for binding to RTE and linking it to the NXF1 export pathway. This protein, the RNA binding motif protein 15 (RBM15), had no previous assigned function. Our analysis revealed direct interaction of RBM15 and a related protein, OTT3, with the essential nuclear export factor NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, have distinct biological roles. These results assign a function to important and conserved mammalian nuclear export factors. These results contribute significantly to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules. We have developed general methods to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Generation of inducible and reversible KO is important for the development of better animal models and for studying the contribution of specific genes in normal and pathologic states.
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