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Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

PURPOSE Bacillus anthracis infection (anthrax) can be highly lethal. Two recent outbreaks related to contaminated mail in the USA and heroin in the UK and Europe and its potential as a bioterrorist weapon have greatly increased concerns over anthrax in the developed world. METHODS This review summarizes the microbiology, pathogenesis, diagnosis, and management of anthrax. RESULTS AND CONCLUSIONS Anthrax, a gram-positive bacterium, has typically been associated with three forms of infection: cutaneous, gastrointestinal, and inhalational. However, the anthrax outbreak among injection drug users has emphasized the importance of what is now considered a fourth disease form (i.e., injectional anthrax) that is characterized by severe soft tissue infection. While cutaneous anthrax is most common, its early stages are distinct and prompt appropriate treatment commonly produces a good outcome. However, early symptoms with the other three disease forms can be nonspecific and mistaken for less lethal conditions. As a result, patients with gastrointestinal, inhalational, or injectional anthrax may have advanced infection at presentation that can be highly lethal. Once anthrax is suspected, the diagnosis can usually be made with gram stain and culture from blood or tissue followed by confirmatory testing (e.g., PCR). While antibiotics are the mainstay of anthrax treatment, use of adjunctive therapies such as anthrax toxin antagonists are a consideration. Prompt surgical therapy appears to be important for successful management of injectional anthrax.