Applications of CRISPR/Cas9 for Selective Sequencing and Clinical Diagnostics

In this chapter, we will discuss the applications of CRISPR/Cas9 in the context of clinical diagnostics. We will provide an overview of existing methods and their use cases in the diagnostic field. Special attention will be given to selective sequencing approaches using third-generation sequencing and PAM-site requirements. As target sequences in an AT-rich environment cannot easily be accessed by the commercially available SpCas9 due to rarity of NGG PAM-sites, new enzymes such as ScCas9 with PAM-site requirements of NNG will be highlighted. Original research on CRISPR/Cas9 systems to determine molecular glioma markers by enriching regions of interest will be discussed in the context of potential future applications in clinical diagnostics

Analysen zur Expression der Deiodinasen und der Hypophysenhormone in der athyroiden Pax8-/- Maus

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CTGF expression during mouse embryonic development

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells

Athyroid Pax8-/- mice cannot be rescued by the inactivation of thyroid hormone receptor α1

The Pax8-/- mouse provides an ideal animal model to study the consequences of congenital hypothyroidism, because its only known defect is the absence of thyroid follicular cells. Pax8-/- mice are, therefore, completely athyroid in postnatal life and die around weaning unless they are substituted with thyroid hormones. As reported recently, Pax8-/- mice can also be rescued and survive to adulthood by the additional elimination of the entire thyroid hormone receptor α (TRα) gene, yielding Pax8 -/-TRαo/o double-knockout animals. This observation has led to the hypothesis that unliganded TRα1 might be responsible for the lethal phenotype observed in Pax8-/- animals. In this study we report the generation of Pax8-/- TRα1-/- double-knockout mice that still express the non-T3-binding TR isoforms α2 and Δα2. These animals closely resemble the phenotype of Pax8-/- mice, including growth retardation and a completely distorted appearance of the pituitary with thyrotroph hyperplasia and hypertrophy, extremely high TSH mRNA levels, reduced GH mRNA expression, and the almost complete absence of lactotrophs. Like Pax8-/- mice, Pax8-/-TRα1-/- compound mutants die around weaning unless they are substituted with thyroid hormones. These findings do not support the previous interpretation that the short life span of Pax8-/- mice is due to the negative effects of the TRα1 aporeceptor, but, rather, suggest a more complex mechanism involving TRα2 and an unliganded TR isoform. Copyrigh

Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling

Pituitary function has been shown to be regulated by an increasing number of intra-pituitary factors including cytokines. Here we show that the important cytokine TNF-α activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-α in GH3 cells was confirmed using real time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-α, western blot analysis showed rapid IκBα degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence labelled p65 and IκBα fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localisation and confirmed IκBα degradation. This was associated with increased NF-κB mediated transcription from an NF-κB responsive luciferase reporter construct. Disruption of NF-κB signaling by expression of dominant negative variants of IKKs or truncated IκBα abolished TNF-α activation of the prolactin promoter, suggesting that this effect was mediated by NF-κB. TNF-α signaling was found to interact with other endocrine signals to regulate prolactin gene expression, and is likely to be a major paracrine modulator of lactotroph function

Dynamic analysis of stochastic transcription cycles

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli

Dynamic organisation of prolactin gene expression in living pituitary tissue

Gene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP). Prolactin promoter activity in transgenic pituitaries varied with time across different regions of the gland. Although amplitude of transcriptional responses differed, all regions of the gland displayed similar overall patterns of reporter gene expression over a 50-hour period, implying overall co-ordination of cellular behaviour. By contrast, enzymatically dispersed pituitary cell cultures showed unsynchronised fluctuations of promoter activity amongst different cells, suggesting that transcriptional patterns were constrained by tissue architecture. Short-term, high resolution, single cell analyses in prolactin-d2EGFP transgenic pituitary slice preparations showed varying transcriptional patterns with little correlation between adjacent cells. Together, these data suggest that pituitary tissue comprises a series of cell ensembles, which individually display a variety of patterns of short-term stochastic behaviour, but together yield long-range and long-term coordinated behaviour