Browsing by Subject "Zinc Fingers"
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Item Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the HTERT Locus(2011-02-01T19:36:40Z) Wilson, Kimberly Anne; Porteus, MatthewThe ability to direct hTERT expression through genetic control or tunable regulatory factors would advance our understanding of the transcriptional regulation of hTERT, and also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and zinc finger nucleases (ZFNs) to target sequences at the hTERT promoter. We first explored expansions to the repertoire of sites that can be targeted by ZFNs and modifications of ZFN architecture to accommodate such sites. A ZFN is made of a zinc-finger DNA binding domain (ZFP) linked to the FokI nuclease domain by a short amino acid “inter-domain linker”. The general sequence motif of a ZFN target is 5’-(ZFN site1)-(6 bp spacer)-(ZFN site2)-3’ and each half-site is 5’-GNNGNNGNN-3’. Variations of this motif come in the forms of variable spacer lengths, extra basepairs in-between triplets, and the inclusion of non-GNN triplets. To explore these types of target sites, we created ZFN variants that contained different inter-domain linkers, lengthened inter-finger linkers, and DNA binding domains created through hybridizing the modular assembly and OPEN methodologies. We show that through altering ZFN architecture, target sites with 5-7-bp spacers and those with ANN, CNN, and TNN triplets can be efficiently recognized and cut by ZFNs. We then generated new ZFPs to five ZFN target sites with 5- or 6-bp spacers in the hTERT locus based on those findings and made ZFTFs by linking the ZFPs to the VP16 transcriptional activation domain. We were able to identify several active ZFTFs that demonstrate a dose-dependent response. The same ZFPs were also converted into ZFNs and screened in combinatorial pairs in cell-based single-strand annealing assays and gene targeting assays. These screening strategies have pinpointed several ZFN pairs that may be useful in genomic editing of the hTERT locus. Our findings provide guidelines for modifying ZFP architecture to a wider array of potential target sites for use in developing ZFTFs and ZFNs at the hTERT promoter, which may be applicable towards inheritable, telomerase-based diseases and answering basic science questions about hTERT transcriptional regulation.Item GATA Co-Factors : Collaborators in Cardiac Development, Conspirators in Cardiac Disease(2005-05-03) Kathiriya, Irfan S.; Srivastava, DeepakDisruption of fetal gene expression during cardiac development can result in congenital heart defects (CHDs), the most common developmental anomaly in humans and the leading non-infectious cause of death in newborns. The reactivation of fetal gene expression during cardiac hypertrophy in the adult is an adaptive response to pressure or volume overloads, but can lead to impaired cardiac function. Gata4, a member of a family of zinc-finger transcription factors, has been implicated as a key regulator of fetal cardiac gene expression during cardiac development and cardiac hypertrophy. This thesis work presents two novel transcriptional complexes likely found in these settings, one that cooperates to activate GATA-dependent transactivation and one that represses it.Item Gene Targeting in a Novel Mouse Model and the Chicken DT40 Cell Line(2010-05-14) Connelly, Jon Patrick; Porteus, MatthewGene targeting has the power to create precise changes at specific sites within the genome. In the context of gene therapy, this technology may be used to treat patients with monogenic diseases by fixing mutations in disease causing genes, followed by transplanting the corrected cells back into the patient. However, the natural rate of gene targeting is too low to be of practical use in most cells; an exception to this is the chicken DT40 cell line which has a high relative rate of gene targeting (gene targeting rate/ random integration rate). We therefore sought to determine the basis of this high rate of gene targeting using assays which quantitate the rates of repairing DNA double-strand breaks through different repair pathways. We show that compared to other cell types, DT40 cells are deficient in random integration. Furthermore, we show this deficiency is due to a reduced ability to repair DNA breaks lacking homology at the ends. In other cell types, the naturally low rate of gene targeting can be stimulated 30-40,000 fold by inducing a double-strand break at the target site. These breaks can be created by proteins called zinc finger nucleases (ZFNs). ZFN mediated gene targeting is a powerful technology, but has not yet been fully characterized in primary cells. Furthermore, before clinical use in the treatment of monogenic diseases, it is necessary to first test this technology in animal models. In the second portion of this dissertation, we developed a mouse model of a generic recessive genetic disease. This model allows the study of gene targeting in any cell population isolated from the mouse. Using this model, we demonstrate ZFN mediated gene targeting in variety of primary cells isolated from the mouse, including ES cells, fibroblasts, and astrocytes. We further demonstrate that targeted stem cells retain their pluripotency, and show that targeted fibroblasts can be transplanted back into a recipient and continue to express protein from the corrected gene. This body of work contributes to bringing the technology of gene targeting closer to clinical application by detailing methods which can be used to further increase gene targeting rates, as well as providing a paradigm in which to study gene targeting followed by transplantationItem Optimizing Zinc Finger Nucleases for Use in Mammalian Cells(2009-01-14) Pruett-Miller, Shondra M.; Porteus, Matthew H.Homologous recombination is a well-established technique that has been used to manipulate the genomes of multiple model organisms with great precision and is therefore being explored as a potential way of performing gene therapy. However, the spontaneous rate of homologous recombination in human cells is too low (10-6) to be therapeutically useful. The most powerful way of stimulating homologous recombination is by introducing a double strand break within the target locus. Zinc Finger Nucleases (ZFNs) are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease and have been used to induce double strand breaks at precise sequences. Although ZFNs have been successfully used to stimulate gene targeting at specific loci, several issues remain. First, a generalized optimal design strategy for making effective and safe ZFNs has yet to be established. Second, a systematic evaluation method needs to be established in which novel ZFNs are evaluated for both functionality and safety. We compare the gene targeting efficiencies and cytotoxicity of ZFNs made by the two established design strategies: modular assembly and a bacterial 2-hybrid selection strategy. We have found that ZFNs made by the bacterial 2-hybrid strategy are both more efficient at stimulating gene targeting and less toxic than ZFNs made by modular assembly. We have also found that ZFNs made via the bacterial 2-hybrid strategy are more efficient at gene targeting using a GFP reporter assay and show less cytotoxicity than previously published 4-finger proteins. We also present a generalized strategy for systematically evaluating new ZFNs, which includes a bacterial (-galactosidase transcription assay, a mammalian gene targeting assay, a mammalian flow cytometry based survival assay, and a 53BP1 foci formation assay. These assays provide a standard for future ZFN design and evaluation, particularly those that may be destined for therapeutic use. Because the issue of toxicity is such and important one, we have also developed possible strategies to reduce toxicity of ZFNs. We will discuss two strategies for regulating ZFN protein expression using small molecules. We show that by regulating protein expression to create ZFNs with shortened half-lives, we can maintain high rates of ZFN mediated gene targeting while reducing ZFN toxicity.