Use of the viral 2A peptide for bicistronic expression in transgenic mice

<p>Abstract</p> <p>Background</p> <p>Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.</p> <p>Results</p> <p>To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.</p> <p>Conclusion</p> <p>The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.</p

Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick

Background The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. Results Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. Results One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.</p

Using individual-level contextual indicators to identify disadvantaged applicants: Evidence from the Foundation Year at Lady Margaret Hall, a college of Oxford University

This provides a working example of how individual-level contextual indicators could be used to target widening-participation interventions specifically for students from low socioeconomic status (low-SES) groups. It is based on the experience of developing selection criteria to identify disadvantaged students for a Foundation Year at Lady Margaret Hall (LMH), a college of Oxford University. The process built on the body of published academic work looking at the most suitable contextual indicators, and the outcome offers a case study of how suitable individual-level indicators can be effectively collected and verified

Using individual-level contextual indicators to identify disadvantaged applicants: Evidence from the Foundation Year at Lady Margaret Hall, a college of Oxford University

This provides a working example of how individual-level contextual indicators could be used to target widening-participation interventions specifically for students from low socioeconomic status (low-SES) groups. It is based on the experience of developing selection criteria to identify disadvantaged students for a Foundation Year at Lady Margaret Hall (LMH), a college of Oxford University. The process built on the body of published academic work looking at the most suitable contextual indicators, and the outcome offers a case study of how suitable individual-level indicators can be effectively collected and verified. Data from applications made to the LMH Foundation Year 2017-2019 showing interaction between individual-level and area-level contextual indicators of disadvantage

The developmental and evolutionary roles of isoforms of regulator of G protein signalling 3 in neuronal differentiation

Fundamental to the complexity of the nervous system is the precise regulation in space and time of the production, maturation, and migration of neurons in the developing embryo. This is eloquently seen in the forming cranial sensory ganglia (CSG) of the peripheral nervous system. Placodes, which are transient pseudostratified neuroepithelia in the surface ectoderm of the embryo, are responsible for generating most of the neurons of the CSG. Placodal progenitors commit to the neuronal fate and delaminate from the epithelium as immature, multipolar neuroblasts. These neuroblasts reside in a staging area immediately outside the placode. Differentiation of the neuroblasts is intimately coupled to their adoption of a bipolar morphology and migration away from the staging area to the future site of the CSG. Thus the forming CSG is a highly tractable model to anatomically separate the three phases of a neuroblast’s lifetime: from neuroepithelial progenitor (in the placode), to immature neuroblast (in the staging area), to mature neuron (in the migratory stream). In this thesis, I used the forming CSG as a model to investigate the role of Regulator of G protein Signalling 3 (RGS3) in neuroblast commitment and differentiation. Promoters within introns of the RGS3 locus generate isoforms in which N-terminal sequences are sequentially truncated, but C-terminal sequences are preserved. Intriguingly, I found that expression of these isoforms in the forming CSG is temporally co-linear with their genomic orientation: longer isoforms are exclusively expressed in the progenitor placode; a medium isoform is expressed exclusively in the neuroblast staging area; and the shortest isoforms are expressed in the neuronal migratory stream. Furthermore, through loss- and gain-of-function experiments, I demonstrated that each of these isoforms plays a specific role in the differentiation state in which it is expressed: placode-expressed isoforms negatively regulate neurogenesis; the neuroblast-expressed isoform negatively regulates differentiation; and the neuron-expressed isoforms negatively regulate neuronal migration. The negative regulatory role which all isoforms play in different cell-biological contexts is intriguing in light of the fact that they all share a C-terminal RGS domain, which canonically negatively regulates G protein signalling. Through domain mutation and deletion, I showed that the RGS and N-terminal domains are important for the function of each isoform. Thus temporally co-linear expression within the RGS3 locus generates later-expressed isoforms which lack the regulatory N-terminal domains of the earlier-expressed isoforms, giving them new license to perform different biochemical functions. Lastly, I investigated the conservation and evolution of RGS3 and its isoforms. RGS3 was found to be present in all extant metazoans, and results from this thesis implicate it as the founding member of the R4 subfamily of RGS proteins. Furthermore, in the early vertebrate lineage, a critical domain was lost. This is intriguing in light of the fact that placodes in their stereotypic forms also emerged early in the vertebrate lineage. Ectopic overexpression of the full-length invertebrate RGS3 protein prevented pseudostratification of the vertebrate placode, suggesting that the domain loss in the early vertebrate lineage was important for the evolution of pseudostratified placodes and the expansion of the vertebrate nervous system. In summary, the work in this thesis has uncovered a previously unseen model of transcriptional regulation of a single locus: intragenic temporal co-linearity. Furthermore, the demonstrated functions of this regulation have profound implications on the generation and differentiation of vertebrate neurons, as well as the evolution of the vertebrate nervous system.This thesis is not currently available in ORA

The IgSF protein MDGA1 regulates morphology during a defined stage of placode-derived neuron maturation in developing chick cranial sensory ganglia.

The developing distal cranial sensory ganglia of the chick present an interesting and tractable model for the study of general processes of neural development. While the early stages of placodal neurogenesis, including induction of the placodes and initiation of the neurogenic programme, have been extensively studied, little is known about the molecular mechanisms that regulate migration of placode-derived neuroblasts and their aggregation to form ganglia. These questions have been addressed in the context of the trigeminal ganglion, however it remains unclear whether these principles apply to the epibranchial ganglia, on which the work presented here is focussed. Molecules potentially involved in controlling placodal neuroblast migration in the epibranchial ganglia were identified through a comparative microarray screen carried out in the Begbie lab. A list of candidate genes implicated in a variety of different cellular was validated by determining expression patterns in the region of the epibranchial CSG by in situ hybridisation. These expression patterns showed that different genes were expressed by different populations within the migratory stream. This question was further addressed through the detailed analysis of the expression patterns of a panel of neuronal and neurogenic markers, leading to the finding that placodal neuroblasts appear to sequentially upregulate different groups of genes as they migrate away from the placode. Neuroblasts within the migratory stream can further be subdivided according to cell morphology, which was assessed through high resolution imaging of GFP-labelled placodal cells. Multipolar and bipolar cells were concentrated around two different regions of the migratory stream with multipolar cells localised near the placode and bipolar cells localised closer to the neural tube. Together these findings support the hypothesis that placodal neuroblasts mature as they migrate towards the site of ganglion aggregation. With this detailed description of the system in mind, the question of molecular control was addressed through the functional characterisation of a candidate gene identified in the original microarray screen. MDGA1, a GPI-anchored IgSF molecule that has been implicated in controlling radial migration of cortical neurons, was specifically expressed in the chick CSG at the relevant stages. RNAi-mediated knockdown and overexpression were used to test the function of MDGA1 in migrating placodal neuroblasts. These experiments showed that MDGA1 negatively regulates the formation and extension of neuronal projections in bipolar neuroblasts. With the mechanisms of MDGA1 function relying entirely on protein-protein interactions at the cell-surface, we then set out to identify and characterise potential MDGA1 binding partners. SPR binding experiments carried out in collaboration with the Aricescu lab revealed that MDGA1 interacts with the Neuroligin family of synaptic proteins. Recent evidence has shown that MDGA1 interacts in cis with NLGN2 in rat hippocampal neurons where it disrupts its interaction in trans with Neurexin1. Neuroligins and Neurexins function to stabilise dendritic filopodia by creating trans-synaptic adhesions and recruiting the synaptic apparatus. Having determined that both NLGN2 and NRXN1 are expressed in placode-derived neuroblasts of the CSG, we propose that these molecules play a role in the stabilisation and extension of neuronal projections in this system and that this function in modulated by MDGA1 function.This thesis is not currently available in OR

Cannabinoid receptor, CB1, expression follows neuronal differentiation in the early chick embryo

The role of the CB1 cannabinoid receptor and endocannabinoid signalling has been widely studied in the adult nervous system. However, an emerging body of evidence suggests that the CB1 receptor may also play a role during development. Here we have scrutinized the expression profile of the CB1 receptor from the onset of neurogenesis in the chick embryo. We find that this gene exhibits a dynamic expression pattern that spatially and temporally follows neuronal differentiation in the early embryo