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Browsing by Subject "Genetic engineering"
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Item Adoption of bioengineered crops(2002-05) Fernandez-Cornejo, Jorge; McBride, WilliamReports on the acceptance of genetically-engineered crops by farmers.Item "D" matters in recombinant AAV DNA packaging(Elsevier, 2021) Zhang, Junping; Guo, Ping; Xu, Yinxia; Mulcrone, Patrick L.; Samulski, R. Jude; Xiao, Weidong; Pediatrics, School of MedicineItem Differentiation and characterization of cell types associated with retinal degenerative diseases using human induced pluripotent stem cells(2014-07-31) Gupta, Manav; Meyer, Jason S.; Belecky-Adams, Teri; Randall, Stephen Karl, 1953-Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.Item Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes(Wiley, 2015-05) Estrada, J.; Martens, G.; Li, P.; Adams, A.B.; Newell, K.A.; Ford, M.L.; Butler, J.R.; Sidner, R.A.; Tector, M.; Tector, A.J.; Department of Surgery, IU School of MedicineBackground Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes. Methods The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. Results Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Nonhuman primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased nonhuman primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2. Conclusions Inactivation of the β4GalNT2 gene reduces human and nonhuman primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of nonhuman primates towards GGTA1/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the nonhuman primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.Item Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses(Springer Nature, 2021-06-23) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of MedicineXenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.Item Hyaluronic acid–binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein(Springer, 2020-12) Shi, Shuiliang; Wang, Congrong; Trippel, Stephen B.; Orthopaedic Surgery, School of MedicineChondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair.Item Intrinsic disorder in protein products of newborn genes(2011-10-19) K., S.; Romero, Pedro; Perumal, Narayanan B.; Dunker, KeithThere are many mechanisms for the creation of new genes. In this study, the newborn genes i.e. de novo genes are the genes that are created from scratch. These are created by two mechanisms, polymerization (de novo genes produced from non-coding regions) and overprinting (de novo genes produced from overlapping frames). Rancurel et al has found that de novo genes in overlapping coding regions tend to be more disordered than their ancestral counterparts. It was suggested that it is natural for the newborn genes to be disordered, as it must be very difficult for newborn genes to obtain order at such an early stage, so that the structure is only developed after the evolutionary development. The two hypotheses tested in this study state (1) that genes generated de novo will have a tendency to be disordered, and (2) this tendency is due to a natural inclination of these genes to be disordered at birth. The origin and evolution of some de novo coding regions have been studied in detail. We analyzed genes reported in literature that have been produced de novo; either by overprinting or by polymerization, and their tendency for disorder was evaluated using the VSL2 disorder predictor. The de novo coding regions produced by both ways indeed shows a tendency towards disorder, which supports hypothesis 1. For hypothesis 2 to be tested on a larger dataset the exonic and intronic materials of two human chromosomes were studied and the tendency for disorder was assessed for any new peptide sequence arising from the translation of non-coding sequences arising from introns and exons (overlapping frames). It was shown that the tendency of disorder for protein products of newborn genes arising from introns were not inclined towards being ordered or disordered, but they can become disordered by evolution. The new exonic material created from the existing exons tends to be more disordered when translated, and this tendency does not seem to be dependent upon the disorder content of the original exons. This difference could be a consequence of the fact that the overlapping frames of coding sequences have indirectly been subjected to evolutionary pressure along with the original exon, whereas intronic sequences do not seem to have this constraint, but the exact nature of this discrepancy needs further study to be explained. The tendency of disorder in the existing new exons seems to be higher than the artificial exons (generated in this study). We conclude that the intrinsic disorder in the protein products of de novo genes is selected by the evolution rather than an initial condition. Thus, the newborn genes were not born disordered.Item Novel methods for the generation of genetically engineered animal models(Elsevier, 2023) Cassidy, Annelise; Onal, Melda; Pelletier, Stephane; Medical and Molecular Genetics, School of MedicineGenetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.Item Tip110 Control of HIV-1 Gene Expression and Replication(2011-08-23) Zhao, Weina; He, Johnny J.; Kaplan, Mark H.; Nakshatri, Harikrishna; Yu, Andy Qigui; Takagi, YuichiroTranscription and alternative splicing play important roles in HIV-1 gene expression and replication and mandate complicated but coordinated interactions between the host and the virus. Studies from our group have shown that a HIV-1 Tat-interacting protein of 110 kDa, Tip110 synergies with Tat in Tat-mediated HIV-1 gene transcription and replication. However, the underlying molecular mechanisms were not fully understood and are the focus of the dissertation research. In the study, we first demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. We then showed that Tip110 was detected at the HIV-1 long terminal repeat (LTR) promoter and associated with increased phosphorylation of serine 2 within the RNAPII C-terminal domain (CTD) and increased recruitment of positive transcription elongation factor b (P-TEFb) to the LTR promoter. Consistent with these findings, we demonstrated that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. During these studies, we also found that Tip110 altered HIV-1 mRNA alternative splicing and increased tat mRNA production. Subsequent analysis indicated that Tip110 selectively increased tat exons 1-2 splicing by activating HIV-1 A3 splice site but had no function in tat exons 2-3 splicing. We then showed that the preferential splicing activity of Tip110 resulted from Tip110 complex formation with hnRNP A1 protein, a negative splicing regulator that binds to the ESS2 element within tat exon 2, and as a result, blocked the complex formation of hnRNP A1 with ESS2 and subsequently activated HIV-1 A3 splice site. Taken together, these results show that Tip110 functions to regulate HIV-1 transcription elongation and HIV-1 RNA alternative splicing. These findings not only add to our understanding of Tip110 biology and function but also uncover a new potential target for development of anti-HIV intervention and therapeutic strategies.Item Transcriptional Regulation of Retinal Progenitor Cells Derived from Human Induced Pluripotent Stem Cells.(2013-08-22) Sridhar, Akshayalakshmi; Meyer, Jason S.; Marrs, James; Belecky-Adams, TeriIn order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.