Matching Items (15)

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Germ Cell Specific Safety Switches: Cell Isolation

Description

The process of spermatogenesis, the differentiation of sperm stem cells into spermatozoa, produces a diverse array of descendent cells which express varied morphological and genetic traits throughout their maturation. Beginning

The process of spermatogenesis, the differentiation of sperm stem cells into spermatozoa, produces a diverse array of descendent cells which express varied morphological and genetic traits throughout their maturation. Beginning with primordial germ cells, these sperm progenitors experience twelve stages of differentiation before maturation into their final stage. During their differentiation, these cells reside in the seminiferous tubules within the testes. These tubules are surrounded by somatic cells, primarily Sertoli, Leydig, myoid, and epithelial cells. These cells provide the germ cells with necessary signaling proteins for their progression as well as protection from exterior toxins through the formation of the blood-testis barrier (BTB). However, their close association with germ cells makes extracting these sperm progenitors difficult. Here, I convey the results for an initial trial of harvesting germ cells from two mice. Due to inconclusive qRT-PCR amplification data from the first experiment, future iterations of this harvest will explore other previously published methods. These will include Magnetic-Activated Cell Sorting which will target individual sperm progenitor populations using cell-surface receptors such as GFRα-1 and THY1 to obtain sperm stem cells. Additionally, Fluorescence-Activated Cell Sorting may be useful for obtaining multiple groups of meiotic cell types from a heterogenous cell suspension harvested from the seminiferous tubules through the use of Hoechst 33342 staining. Finally, extraction of spermatozoa from the Cauda Epididymis, a storage site for these mature sperm, can be performed either in conjunction with testes collection during necropsy or as an in vivo technique intended for serial sampling of sperm cells over time. Regardless, it is necessary for these methods to produce populations from spermatogonia to spermatozoa with high purity in order to produce representative qRT-PCR results downstream, indicating either presence or lack of genetic mutation enacted by future CRISPR-Cas9 experiments.

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  • 2019-05

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CRISPR Based Synthetic Transcription Factors: The Future of Transcriptional Therapeutics

Description

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR based transcriptional regulators have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. However, for this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Here, we discuss recent advances and integrative strategies that can help pave the way towards a new class of transcriptional therapeutics.

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  • 2019-05

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Engineering Self-Organizing Biliary Organoids from Human Induced Pluripotent Stem Cells

Description

Cholangiocytes, the epithelial cells of the bile duct, are the origin of cholangiopathies which often necessitate liver transplants. Current progress in generating functional biliary organoids show potential for modelling cholangiopathies

Cholangiocytes, the epithelial cells of the bile duct, are the origin of cholangiopathies which often necessitate liver transplants. Current progress in generating functional biliary organoids show potential for modelling cholangiopathies and validating therapeutic drugs. Organoids by groups Ogawa et al. and Sampaziotis et al. utilize soluble molecule induction, OP9 co-culture, and three-dimensional culture to achieve self-organizing tissues which express mature cholangiocyte markers and show cholangiocyte functionality. This thesis describes our efforts to establish a standard for functional PSC-derived bile duct tissues. By directing cell fate and patterning through external cues alone, we were able to produce CK19+ALB+ hepatoblast-like cells. These soluble molecule-induced cells also expressed EpCAM and CEBPA, suggesting the presence of early liver epithelial cells. However, inconsistent results and high levels of cell death with soluble molecule induction in early stages of differentiation prompted the development of a combinatory differentiation method which utilized multiple differentiation tools. We opted to combine transcription-factor triggered differentiation with soluble molecule-mediated differentiation to produce early biliary cells with the potential to develop into mature cholangiocytes. By combining genetic engineering through the activation of doxycycline-inducible GATA6 switch and microbead-mediated CXCR4 separation, we generated patterned tissues which expressed early biliary markers, CD146, CK19, and SOX9. In the future, three-dimensional cell culture and OP9 co-culture could improve our current results by facilitating 3D cellular self-organization and promoting NOTCH signaling for cholangiocyte maturation. Next steps for this research include optimizing media formulations, tracking gene expression over time, and testing the functionality of generated tissues.

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Date Created
  • 2017-05

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Safe CRISPR: Challenges and Opportunities

Description

Conservatism is intrinsic to safety of emerging biotechnologies. Fear of unintended consequences, misuse, and bioterror are rightfully essential in our discussions of novel innovations. Clustered regularly Interspaced Short Palindromic Repeats

Conservatism is intrinsic to safety of emerging biotechnologies. Fear of unintended consequences, misuse, and bioterror are rightfully essential in our discussions of novel innovations. Clustered regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins are no exception. This review will characterize environmental and health-related risks of CRISPR-applications and expound upon mechanisms that are or can be used to minimize risk. CRISPR is broadening access and simplifying genomic and transcriptomic editing leading to wide-range usage in all of biology. Utilization in gene therapies, gene drives, and agriculture could all be universally impactful applications that need their own safety technologies and guidelines. The initial ethical guidelines and recommendations, that will guide these technologies, are being steadily developed. However, technical advances are required to facilitate safe usage. Since the advent of CRISPR gene editing in 2012 advances to limit off-target edits (both cellular and genomic) have been developed. Delivery systems that use viral or nanoparticle packaging incorporate safety mechanisms to guard against undesirable side effects are being produced and rigorously tested. Besides its applications in basic biology and potential as a gene therapy, CRISPR had humbler beginnings. Industrially it was, albeit unknowingly, used to fend off infection in productions of yogurt batches. This was one of the earliest applications of CRISPR, however with the knowledge we now have ecological and industrial uses of CRISPR have multiplied. Gene drives have the power to spread genetic mutations throughout populations and agricultural uses to better crop genomes are also of interest. These uses have struck a chord with interest groups (environmentalists, anti-GMO groups, etc) who imagine how this technology can drastically alter species with unforeseen evolutionary changes that could reshape present-day ecosystems. This review will describe existing technologies that will safeguard humanity and its interests while fully employing CRISPRs far-reaching potentiality.

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Date Created
  • 2018-05

CRISPR/Cas9 Mediated Mutation in the ATP-ase Domain of XPB to Study its Role in Pancreatic Ductal Adenocarcinoma

Description

Pancreatic ductal adenocarcinoma (PDAC) is a form of pancreatic cancer that affects the exocrine function of the pancreas. PDAC is often hard to diagnose and has shown to also be

Pancreatic ductal adenocarcinoma (PDAC) is a form of pancreatic cancer that affects the exocrine function of the pancreas. PDAC is often hard to diagnose and has shown to also be as difficult to treat. Xeroderma pigmentosum type B (XPB), is a protein can be found in Transcription Factor II Human (TFIIH). It is known to have ATP-ase and helicase activities. The ATP-ase activities could be used to regulate the transcription within super enhancer (SE) networks. Knocking out the ATP-ase activity in XPB in the same way that triptolide does would offer a more individualized therapeutic regiment. A loss of function mutation was tested to identify whether or not the mutation was present within the strand of DNA. In order to explore the role of XPB in pancreatic cancer, a knockout clone was made through the use of the CRISPR/Cas9 genome editing technology to induce a clone in exon 2 of XPB using a plasmid with Green Fluorescent Protein (GFP) selection marker. Once the clones were successfully made, they underwent testing through the use of a Surveyor Mutation Detection Kit for standard electrophoresis. The confirmation of a functional clone lead to GFP, which contained the mutation, being chosen for further testing be compared to the wild type GFP. After the GFP D54H mutation was chosen for further testing, it was then cultured from bacteria and wild type GFP and GFP D54H underwent a restriction enzyme digest. The digest resulted in showing that GFP and GFP D54H were the same on a larger level, and that one of the only ways to prove that the mutation was present was through amplification and analysis using the mutation detection kit.

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Date Created
  • 2017-05

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Engineering Open Chromatin with Synthetic Pioneer Factors:: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin

Description

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.

I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.

SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.

Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.

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Date Created
  • 2019

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A rapid lipid-based approach for normalization of quantum dot-detected biomarker expression on extracellular vesicles in complex biological samples

Description

Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status.

Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation.

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Date Created
  • 2019

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Using Bioengineering Approaches to Generate a Three-Dimensional Human Induced Pluripotent Stem-Cell Based Model of Alzheimer's Disease

Description

The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not

The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not allow for the study of the pathophysiology. In vitro models of AD generated with patient derived human induced pluripotent stem cells (hiPSCs) could provide new insight into disease mechanisms. Although many protocols exist to differentiate hiPSCs to neurons, standard practice relies on two-dimensional (2-D) systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. This research aims to create three-dimensional (3-D) models of AD using hiPSCs, which would enhance the understanding of AD pathophysiology thereby, enabling the generation of effective therapeutics.

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Date Created
  • 2017

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Developing safe and controllable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based therapies with design principles of synthetic biology

Description

The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot

The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies.

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Date Created
  • 2020

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Three-Dimensional Microfluidic Based Tumor-Vascular Model to Study Cancer Cell Invasion and Intravasation

Description

Breast cancer is the second leading cause of disease related death in women, contributing over

40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor

understanding of

Breast cancer is the second leading cause of disease related death in women, contributing over

40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor

understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer

metastasis includes the invasion and intravasation that results in cancer cells disseminating from

the primary tumor and colonizing distant organs. However, the integrated study of invasion and

intravasation has proven to be challenging due to the difficulties in establishing a combined tumor

and vascular microenvironments. Compared to traditional in vitro assays, microfluidic models

enable spatial organization of 3D cell-laden and/or acellular matrices to better mimic human

physiology. Thus, microfluidics can be leveraged to model complex steps of metastasis. The

fundamental aim of this thesis was to develop a three-dimensional microfluidic model to study the

mechanism through which breast cancer cells invade the surrounding stroma and intravasate into

outerlying blood vessels, with a primary focus on evaluating cancer cell motility and vascular

function in response to biochemical cues.

A novel concentric three-layer microfluidic device was developed, which allowed for

simultaneous observation of tumor formation, vascular network maturation, and cancer cell

invasion/intravasation. Initially, MDA-MB-231 disseminated from the primary tumor and invaded

the acellular collagen present in the adjacent second layer. The presence of an endothelial network

in the third layer of the device drastically increased cancer cell invasion. Furthermore, by day 6 of

culture, cancer cells could be visually observed intravasating into the vascular network.

Additionally, the effect of tumor cells on the formation of the surrounding microvascular network

within the vascular layer was evaluated. Results indicated that the presence of the tumor

significantly reduced vessel diameter and increased permeability, which correlates with prior in vivo

data. The novel three-layer platform mimicked the in vivo spatial organization of the tumor and its

surrounding vasculature, which enabled investigations of cell-cell interactions during cancer

invasion and intravasation. This approach will provide insight into the cascade of events leading up

to intravasation, which could provide a basis for developing more effective therapeutics.

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Date Created
  • 2017