Browsing by Subject "TALEN"
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Item Application of targeting nucleases to modeling prostate cancer gene rearrangements(2014-06) Nyquist, Michael D.Advanced prostate cancer (PCa) treated with androgen deprivation therapy (ADT) eventually relapses to an ADT-resistant disease referred to as castration resistant PCa (CRPC). Recent integrative analyses of PCa genomes have led to the elucidation of potential subtypes that are revelatory to the development of PCa as well as the mechanisms of resistance to ADT and CRPC progression. These studies have confirmed that alterations in the androgen receptor (AR) signaling axis are central to CRPC progression, and have uncovered complex mechanisms by which AR and other components of the AR signaling axis affect, and are affected by, genomic changes and epigenetic transformations. Among the most frequent alterations in CRPC are direct alterations in the AR gene. These AR gene alterations include AR amplification, point mutations, and more recently AR gene rearrangements leading to expression of truncated, constitutively active AR splice variants that are impervious to ADT. Fortunately, the recent development of transcription activator-like effector nucleases (TALENs) has allowed researchers to tailor the genomes of their model systems more rigorously than ever before. This dissertation presents studies centered on genome engineered cell lines modeling intragenic-AR rearrangements that are associated with the production of androgen receptor splice variants. In the second chapter, two AR rearrangements associated with ARv567es expression were recreated in PCa cell line R1-AD1 using targeting nucleases. These engineered cell lines expressed high levels of Arv567es that recapitulated the full length AR transcriptome and drove androgen independent growth. In chapter three, AR rearrangements associated with high AR-V7 expression in CRPC cell lines CWR-R1 and 22Rv1 were induced and gene-corrected, respectively, using targeting nucleases. We found that a deletion contained in AR intron 1 of CWR-R1 was not sufficient to induce AR-V7 expression. However, genetic correction of a duplicated AR intron 3 region found in 22Rv1 decreased AR-V7 levels. These models were developed to determine the underlying mechanisms of AR variant production as well as provide a novel platform with which to study AR variant DNA binding, transcriptional regulation, and clinically relevant aspects of PCa such as biomarker research and precision medicine.Item Genome engineering in large animals for agricultural and biomedical applications(2013-08) Tan, WenfangPrecision genetics will enhance genome-based improvement of livestock for agriculture and biomedicine. This thesis aimed to modify large animal genomes with precision; as the technologies progressed, our capability expanded from random insertional transgenesis to nucleotide-level precision. It began with Sleeping Beauty (SB) transposon mediated rapid integration of dominant negative Myostatin alleles. All piglets generated from treated cells harbored the transgenes; however, we were unable to study phenotypes due to death of the founder animals. We then sought to introgress a SNP into porcine Myostatin through recombinant Adeno-associated Virus (rAAV) mediated gene targeting. We achieved a 2x10-4 targeting frequency but only one-half of the targeted colonies harbored the SNP. Similarly, we succeeded in porcine LDLR gene knockout; however, targeted clones were often confounded by "bystander" cells with only random insertions of the targeting vector. We turned to develop TALENs for efficient targeting of important genes. TALENs demonstrated high activity in both cultured primary fibroblasts and early stage embryos. A simple SB transposon based co-selection strategy enabled enrichment for TALEN modified cells and efficient isolation of modified clones: single gene mono- and bi-allelic modification was induced in up to 54% and 17% of colonies respectively. It also enabled isolation of colonies harboring large chromosomal deletions (10% of colonies) and inversions (4%) after treatment with two TALEN pairs. We derived miniature swine models of familial hypercholesterolemia from LDLR mono- and bi-allelic TALEN-knockout fibroblasts. We next utilized TALEN and CRISPR/Cas9 stimulated homology-directed repair (HDR) to edit genes with oligonucleotide, plasmid, and rAAV templates without any drug selection. We first introgressed a bovine POLLED allele into horned dairy bull fibroblasts to circumvent manual dehorning. We also introduced single-nucleotide alterations or small indels into 14 additional genes in pig, cattle and sheep, into 10-50% of cells from fibroblast populations treated with TALEN mRNA and oligonucleotides. Up to 67% of propagated colonies harbored the intended edits and over one-half were homozygous. Some edits were naturally occurring SNP alleles, equivalent to non-meiotic inter- or intra-species introgression of valuable alleles. We created pig models for infertility and colon cancer from colonies with TALEN-HDR knockout alleles in DAZL and APC.