Toward group II intron-based genome targeting in eukaryotic cells

dc.contributor.advisorLambowitz, Alanen
dc.creatorVernon, Jamie Leeen
dc.description.abstractMobile group II introns consist of a self-splicing RNA molecule and an intron-encoded protein with reverse transcriptase activity that function together in an RNP and catalyze the insertion of the intron into specific DNA target sites by a process known as retrohoming. The mechanism of insertion requires the intron RNA to bind and reverse splice into one strand of the DNA target site, while the intron-associated protein cleaves the opposite DNA strand and reverse transcribes the intron RNA. DNA target site recognition and binding are dependent upon base pairing between the intron RNA and the target DNA molecule. By modifying the recognition sequences in the intron RNA, group II introns can be engineered to insert into virtually any desired target DNA. Based on this technology, a novel class of commercially available group II intron-based gene targeting vectors, called targetrons, has been developed. Targetrons have been used successfully for gene targeting in a broad range of bacteria. Previously, our laboratory demonstrated that group II introns retain controllable retrohoming activity in mammalian cells, albeit with very low targeting efficiency. However, the gene targeting capability of group II introns is not limited to direct insertion of the intron. Group II introns can also create double-strand breaks that stimulate homologous recombination. By virtue of these attributes, mobile group II introns offer great promise for applications in genetic engineering, functional genomics and gene therapy. Here I present the results of experiments in which I tested group II introns for gene targeting activities in eukaryotic cells. First, I demonstrated that group II introns injected into zebrafish (Danio rerio) embryos retain in vivo plasmid targeting activity that is enhanced by the addition of magnesium chloride and deoxynucleotides. I also verified that similar in vivo targeting activity is retained in Drosophila melanogaster embryos. Further, I describe repeated experiments in zebrafish embryos designed to target the zebrafish genome with inconclusive results. Group II introns were also delivered to cultured human cells for genome targeting. Here I present promising evidence for the ability of group II introns to stimulate homologous recombination between an exogenously introduced donor DNA molecule and the chromosome. The donor DNA was delivered either as a linearized double-stranded plasmid by electroporation or as a single stranded genome of a recombinant adeno-associated virus (AAV). In both cases, cells receiving both the group II intron RNP and the donor DNA showed more efficient integration of the donor DNA than introduction of the donor DNA alone. The studies presented here provide insight into the potential of using group II introns for future applications in gene targeting in eukaryotes.en
dc.description.departmentInstitute for Cellular and Molecular Biologyen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subjectGene targetingen
dc.subjectGenome targetingen
dc.subjectGroup II intronsen
dc.subjectEukaryotic cellsen
dc.titleToward group II intron-based genome targeting in eukaryotic cellsen and Molecular Biology, Institute foren and Molecular Biologyen University of Texas at Austinen of Philosophyen

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