Gene Targeting in Eukaryotic Cells by Purified Group II Introns
Background Functional genomic disciplines seek to understand the contributions of genes and their protein products in both 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 principal 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. Manners to apply these technologies in eukaryotic cells are limited.
Invention Description Group II Intron Ll.LtrB is a mobile element that can be used to target essentially any gene in cells of various origins. Successful expression of active group II intron RNA and protein components in bacteria produces ribonucleoprotein (RNP) particles that can be purified and used for various gene targeting experiments. Purified RNPs can be injected, electroporated, or transfected into eukaryotic cells including mammalian cells, zebrafish, and Xenopus laevis oocyte nuclei. Once injected, the RNPs deliver a sequence that can integrate into the target sequence. Homologous recombination produces an insertion event that can be used to knock out known genes, to insert a new gene into a chromosome, or to insert a reporter gene into a random site in the chromosome to identify regulatory sequences. These tools will make important contributions to functional genomics and the study of genes related to disease conditions.
Can be used to target any gene in cells of various origin Useful tool for functional genomics studies
Group II intron RNPs can establish stable integration of DNA fragment at specific loci. Methods to purify group II intron RNPs from E. coli cells are established. RNPs can be injected, electroporated, or transfected.
IP Status One foreign patent application filed
UT Researcher Alan M. Lambowitz, Ph.D., Chemistry and Biochemistry, The University of Texas at Austin Xiaoxia Cui, Ph.D., School of Biological Sciences, The University of Texas at Austin Roland J. Saldanha, Ph.D., School of Biological Sciences, The University of Texas at Austin Jamie L. Vernon, M.S., The University of Texas at Austin Kazuo Watanabe, Ph.D., School of Biological Sciences, The University of Texas at Austin Marta Mastroianni, M.S., School of Biological Sciences, The University of Texas at Austin
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