Recombinant protein expression by targeting pre-selected chromosomal loci

<p>Abstract</p> <p>Background</p> <p>Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.</p> <p>Results</p> <p>We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.</p> <p>Conclusion</p> <p>RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.</p

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier

Neither right nor wrong?:Ethics of collaboration in transformative research for sustainable futures

Transformative research is a broad and loosely connected family of research disciplines and approaches, with the explicit normative ambition to fundamentally question the status quo, change the dominant structures, and support just sustainability transitions by working collaboratively with society. When engaging in such science-practice collaborations for transformative change in society, researchers experience ethical dilemmas. Amongst others, they must decide, what is worthwhile to be researched, whose reality is privileged, and whose knowledge is included. Yet, current institutionalised ethical standards, which largely follow the tradition of medical ethics, are insufficient to guide transformative researchers in navigating such dilemmas. In addressing this vacuum, the research community has started to develop peer guidance on what constitutes morally good behaviour. These formal and informal guidelines offer a repertoire to explain and justify positions and decisions. However, they are only helpful when they have become a part of researchers’ practical knowledge ‘in situ’. By focusing on situated research practices, the article addresses the need to develop an attitude of leaning into the uncertainty around what morally good behaviour constitutes. It also highlights the significance of combining this attitude with a critical reflexive practice both individually and collaboratively for answering questions around ‘how to’ as well as ‘what is the right thing to do’. Using a collaborative autoethnographic approach, the authors of this paper share their own ethical dilemmas in doing transformative research, discuss those, and relate them to a practical heuristic encompassing axiological, ontological, and epistemological considerations. The aim is to support building practical wisdom for the broader research community about how to navigate ethical questions arising in transformative research practice.</p

Neither right nor wrong?:Ethics of collaboration in transformative research for sustainable futures

Transformative research is a broad and loosely connected family of research disciplines and approaches, with the explicit normative ambition to fundamentally question the status quo, change the dominant structures, and support just sustainability transitions by working collaboratively with society. When engaging in such science-practice collaborations for transformative change in society, researchers experience ethical dilemmas. Amongst others, they must decide, what is worthwhile to be researched, whose reality is privileged, and whose knowledge is included. Yet, current institutionalised ethical standards, which largely follow the tradition of medical ethics, are insufficient to guide transformative researchers in navigating such dilemmas. In addressing this vacuum, the research community has started to develop peer guidance on what constitutes morally good behaviour. These formal and informal guidelines offer a repertoire to explain and justify positions and decisions. However, they are only helpful when they have become a part of researchers’ practical knowledge ‘in situ’. By focusing on situated research practices, the article addresses the need to develop an attitude of leaning into the uncertainty around what morally good behaviour constitutes. It also highlights the significance of combining this attitude with a critical reflexive practice both individually and collaboratively for answering questions around ‘how to’ as well as ‘what is the right thing to do’. Using a collaborative autoethnographic approach, the authors of this paper share their own ethical dilemmas in doing transformative research, discuss those, and relate them to a practical heuristic encompassing axiological, ontological, and epistemological considerations. The aim is to support building practical wisdom for the broader research community about how to navigate ethical questions arising in transformative research practice.</p

More fodder for the oven? Dealing with forest-related conflicts arising from the production and use of energy wood in Europe

Results from the COOL project201

Biodiversity post-2020: Closing the gap between global targets and national-level implementation

National and local governments need to step up efforts to effectively implement the post-2020 global biodiversity framework of the Convention on Biological Diversity to halt and reverse worsening biodiversity trends. Drawing on recent advances in interdisciplinary biodiversity science, we propose a framework for improved implementation by national and subnational governments. First, the identification of actions and the promotion of ownership across stakeholders need to recognize the multiple values of biodiversity and account for remote responsibility. Second, cross-sectorial implementation and mainstreaming should adopt scalable and multifunctional ecosystem restoration approaches and target positive futures for nature and people. Third, assessment of progress and adaptive management can be informed by novel biodiversity monitoring and modeling approaches handling the multidimensionality of biodiversity change