Bone Morphogenetic Protein (BMP) signaling in development and human diseases

Bone Morphogenetic Proteins(BMPs)are a group of signaling molecules that belongs to the Transforming Growth Factor-b(TGF-b)superfamily of proteins.Initially discovered for their ability to induce bone formation,BMPs are now known to play crucial roles in all organ systems.BMPs are important in embryogenesis and development,and also in maintenance of adult tissue homeostasis.Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects,highlighting the essential functions of BMPs.In this review,we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development.A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.

CRISPR clear? Dimeric Cas9-Fok1 nucleases improve genome-editing specificity

Bacteria and archaea have been known for decades having evolved adaptive immunedefenses called clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated(Cas)systems to degrade foreign nucleic acids.Recently,these RNA-guided Cas9 nucleases derived from CRISPR/Cas systems have shown promise in transforming our ability to edit mammalian genomes.1 While zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs)have shown similar promise,the ease of producing targeting RNAs over the generation of unique sequence-directed nucleases to guide site-specific modifications makes the CRISPR/Cas9 system an appealing method forgenome editing.A short guideRNA(sgRNA)can direct Cas9 to a specific genomic sequence where it induces doublestrand breaks that,when imperfectly repaired,yield mutations.1 Cas9 can also catalyze gene replacement through homologous recombination.