The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

"Secretary in Fashion" and "Love-Letters": a study of socio-cultural and pragmatic-linguistic features in the late 17th century English language

Epistolary manuals are conspicuous historical documents for the pedagogy of letter writing; however, their actual usage as manuals by letter writers is unknown. "Secretary in Fashion" by Serre (1668), an epistolary manual, and "Love-Letters between a Nobleman and his Sister" (1684), an epistolary novel attributed to Behn, both give insights into epistolary conventions. Their inception and nature is interesting, considering their historical context. Despite the Restoration of Charles II, 17th century England was in a confused political state; as a result, texts regarding social convention or politics interested contemporary readers (the novel is inspired by a scandal of Lord Grey, an ardent Whig opposing Charles II). Past epistolary studies focus on 18th rather than 17th century manuals; the latter is typically used as supplementary information. Similarly, past epistolary fiction studies focus on 18th century texts; moreover, linguistic studies on Behn and the novel are deficient. Thus, this study addresses the research questions: 1) What are the socio-cultural and pragmaticlinguistic features represented in "Secretary in Fashion"? 2) What are the socio-cultural and pragmatic-linguistic features represented in "Love-Letters between a Nobleman and his Sister", and do any of these features correlate with the features represented in "Secretary in Fashion"? How far do the characteristic linguistic features of "Love-Letters between a Nobleman and his Sister" correlate with the practices recommended by the manual? Both texts were qualitatively analysed from an historical pragmatic perspective, which observes the potential effects of the socio-cultural and historical context. Also, as the texts concern shared discourses, comparisons were made with Gricean and Politeness Theory. The results show that the manual is a typical 17th century epistolary manual, aligning particularly with the "Academies of Complements", as it concerns the social conventions of the gentry. The novel mainly upheld instructions on form and matter; deviations occurred due to the amatory nature of some letters, and the narrative force affecting the style. Unfortunately, neither research question elucidates the actual usage of manuals. However, this study does show the epistolary practices of two writers, within specific contexts. It reveals that their 17th century English language use is affected by socialisation, in terms of social conventions concerning social rank, age, and gender; therefore, context varies language use. Also, their popularity reveals the interests of the 17th century society. Interest in epistolary-related texts, surely piques the interest of the modern reader as to why such epistolary-related texts were interesting.