Methods for Expressing RNP Particles in Eukaryotic Cells
Background Functional genomic disciplines seek to understand the contributions of genes and their protein products in healthy and disease conditions. Due to evolutionary conservation, model organisms provide simplified systems for analysis, while still producing relevant results. Gene function can be accessed by two principle methods ? gain of function and loss of function methodologies. Gain of function approaches seek to express novel genes in organisms or overexpress the corresponding gene in a model organism. Non-targeted insertions of reporter genes can allow the discovery of DNA regulatory elements with desired properties. Loss of function approaches focus on gene disruption. In both approaches, the physiologic consequences of the gene manipulation is measured. Yeast represents a favored eukaryotic genetic system. The group II intron Lactococcus lactis LtrB is a ribozyme that catalyzes its own splicing and is able to insert itself into essentially any gene in different bacterial species. In order to use group II introns to manipulate eukaryotic genomes, the catalytically active ribonucleoprotein (RNP) particles must be introduced into cells. One option is to express the RNP particles in the cells; this will allow targeted or random gene targeting/inactivation events. However, manners to apply these technologies in eukaryotic cells are limited.
Invention Description Successful expression of active group II intron RNA and protein components in yeast allows exploitation of defined and random mutations to determine what factors may be limiting eukaryotic cells. The short life cycle and facile manipulation of yeast expedite the process. Once rate limiting processes are identified, the knowledge can be easily transferred to other eukaryotic systems. Mobile group II intron-encoded protein, LtrA, and the intron RNA have been successfully expressed in mammalian tissue culture cells. The expressed intron-encoded protein containing a nuclear localization signal (NLS) is localized into the nucleus. The intron RNA splices when co-expressed with intron protein indicating that ribonucleoprotein (RNP) particles are produced. The functional RNP particles expressed in vivo are capable of targeting chromosomal genes. In sufficient quantities, RNP particles can be used to disrupt or modify any desired gene, with applications in functional genomics and gene therapy.
Allows for identification and optimization of rate limiting steps in eukaryotic DNA targeting Rapid optimization allowed by the short life cycle and inexpensive culture conditions of yeast
Combination of mobile group II intron RNA and protein components form a RNP capable of genome insertion at desired locations
IP Status One U.S. patent application filed
UT Researcher Alan M. Lambowitz, Ph.D., Chemistry and Biochemistry, The University of Texas at Austin Roland J. Saldanha, Ph.D., School of Biological Sciences, The University of Texas at Austin Fanglei Zhuang, BA, Chemistry and Biochemistry, The University of Texas at Austin
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