Quantitative Comparison of Constitutive Promoters in Human ES cells

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs

NANOG Reporter Cell Lines Generated by Gene Targeting in Human Embryonic Stem Cells

Background: Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency, but the precise role and regulation of NANOG are not well defined. Methodology/Principal Findings: To facilitate the study of NANOG expression and regulation in viable hESC cultures, we generated fluorescent NANOG reporter cell lines by gene targeting in hESCs. In these reporter lines, the fluorescent reporter gene was co-expressed with endogenous NANOG and responded to experimental induction or repression of the NANOG promoter with appropriate changes in expression levels. Furthermore, NANOG reporter lines facilitated the separation of hESC populations based on NANOG expression levels and their subsequent characterization. Gene expression arrays on isolated hESC subpopulations revealed genes with differential expression in NANOG high and NANOG low hESCs, providing candidates for NANOG downstream targets hESCs. Conclusion/Significance: The newly derived NANOG reporter hESC lines present novel tools to visualize NANOG expression in viable hESCs. In future applications, these reporter lines can be used to elucidate the function and regulation of NANO

Full production cycle performance of gene-edited, sterile Atlantic salmon - growth, smoltification, welfare indicators and fillet composition

Using germ cell-free (GCF), sterile, dnd-knockout salmon for farming could solve the problems associated with precocious maturation and genetic introgression of farmed breeds into wild populations. However, prior to using GCF fish in the salmon farming industry, it is crucial to understand if, or how, the GCF phenotype differs from wild type (WT) counterparts in terms of growth and welfare. To characterize the GCF phenotype throughout a production cycle, we reared GCF and WT salmon in indoor common garden tanks for 3 years, until harvest size. Regarding body size, smoltification markers (mRNA levels of gill Na+/K+-ATPase [NKA] subunits), plasma stress indicators (pH, glucose, sodium, chloride, calcium), relative heart size, prevalence of vertebra deformities and fillet proximate composition, GCF fish could not be distinguished from WTs. Transient differences were detected in plasma concentrations of lactate and osmolality, and only a few genes were differentially expressed in WT and GCF transcriptomes of muscle and pituitary. At harvest, fillets from GCF and WT salmon contained the same amount of omega-3 fatty acids, however the relative content of omega-3 fatty acids was higher in GCF compared to WT males. Towards harvest size, body growth rate, condition factor and relative liver size were significantly higher in WT than in GCF fish, probably relating to initiation of puberty in WTs. Since GCF salmon never become sexually mature, it is possible to postpone the time of harvest to exploit the growth potential uninhibited by sexual maturation. In conclusion, GCF salmon performed to a large extent similarly to their WT counterparts but had the clear advantage of never maturing

Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications