Springer protocols feed by Biotechnology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Derivation and Chondrogenic Commitment of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors
The induction of human embryonic stem cells to a mesenchymal-like progenitor population constitutes a developmentally relevant approach for efficient directed differentiation of human embryonic stem (hES) cells to the chondrogenic lineage. The initial enrichment of a hemangioblast intermediate has been shown to yield a replenishable population of highly purified progenitor cells that exhibit the typical mesenchymal stem cell (MSC) surface markers as well as the capacity for multilineage differentiation to bone, fat, and cartilage. Herein, we provide detailed methodologies for the derivation and characterization of potent m...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news
Mesenchymal Stem Cells Derived from Human Adipose Tissue
Human adult mesenchymal stem cells are present in fat tissue, which can be obtained using surgical procedures such as liposuction. The multilineage capacity of mesenchymal stem cells makes them very valuable for cell-based medical therapies. In this chapter, we describe how to isolate mesenchymal stem cells from human adult fat tissue, propagate the cells in culture, and cryopreserve the cells for tissue engineering applications. Flow cytometry methods are also described for identification and characterization of adipose-derived stem cells and for cell sorting. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news
Mesenchymal Stem Cells Derived from Human Bone Marrow
Mesenchymal stem cells are found in a number of tissues and have the potential to differentiate into a range of mesenchymal lineages. This ready availability and multipotent character means that mesenchymal stem cells have become a focus for the field of tissue engineering, particularly for the repair of bone and cartilage. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news
Human Fetal and Adult Chondrocytes
As the only cell type found in healthy adult cartilage, chondrocytes are the obvious and most direct starting point for cartilage tissue engineering. Human adult, juvenile, neonatal, and fetal chondrocytes have all been demonstrated to produce cartilage matrix components in vitro for production of engineered tissues. In this chapter, procedures are outlined for isolation of chondrocytes from human fetal and adult cartilage. Methods for expansion and cryopreservation of the cells and characterization of gene expression using quantitative polymerase chain reaction (Q-PCR) analysis are also described. (Source: Springer protoc...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news
Cartilage Tissue Engineering: What Have We Learned in Practice?
Many technologies that underpin tissue engineering as a research field were developed with the aim of producing functional human cartilage in vitro. Much of our practical experience with three-dimensional cultures, tissue bioreactors, scaffold materials, stem cells, and differentiation protocols was gained using cartilage as a model system. Despite these advances, however, generation of engineered cartilage matrix with the composition, structure, and mechanical properties of mature articular cartilage has not yet been achieved. Currently, the major obstacles to synthesis of clinically useful cartilage constructs are our in...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news
Fluorescence Labeling of Short RNA by Oxidation at the 3′-End
In RNA nanotechnology, construction of nanoparticles involves conjugation of functionalities, cross-linking of modules, labeling of RNA subunits, and chemical modification of nucleotides. Efficiency and sensitivity are important for the RNA labeling, which also can be used as probes in microarrays, Northern blotting, and gel-shift assays. Here, we describe a method for fluorescence labeling of short RNA at the 3′-end by oxidation. The 3′-terminus of in vitro-transcribed short RNA is oxidized by sodium periodate, and fluorescein-5-thiosemicarbazide is added after removal of excess oxidant. Purified short RNA wit...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Functional Assays for Specific Targeting and Delivery of RNA Nanoparticles to Brain Tumor
Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miR...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Aptamer-Mediated Nanoparticle Interactions: From Oligonucleotide–Protein Complexes to SELEX Screens
Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They can be easily immobilized on various surfaces such as nanoparticles. Functionalized particles can then be used to various aims. We took advantage of the AlphaScreen® technology for monitoring aptamer-mediated interactions. A particle bearing an aptamer contains a photosensitizer whereas another type of particle contains a chemiluminescer. Irradiation causes the formation of singlet oxygen species in the photosensitizer-containing bead that in turn activates the chemiluminescer. Luminescence emission can be observed if ...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
A High-Throughput Screening Assay for the Functional Delivery of Splice-Switching Oligonucleotides in Human Melanoma Cells
Since the conception of RNA nanotechnology (Cell, 94:147, 1998), there has been tremendous interest in its application for the functional delivery of RNA into cells. Splice-switching oligonucleotides (SSOs) are an emerging antisense drug class with the ability to therapeutically modify gene expression. A wide variety of chemical modifications have been devised to try to increase the activity and stability of SSOs. Also, as with most nucleic acid therapeutics, delivery into the cell is the major hurdle for in vivo and clinical applications. As a result, various RNA nanoparticles are being constructed for targeted delivery o...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Methods for Assembling B-Cell Lymphoma Specific and Internalizing Aptamer–siRNA Nanoparticles Via the Sticky Bridge
Structured functional RNA entities, including aptamers and siRNAs, have amazing versatility in structure and function. These molecules can serve as powerful, attractive building blocks for the bottom-up assembly of complex nanostructures. Here, we describe novel cell-type specific and internalizing B-cell activating factor receptor (BAFF-R) aptamer–siRNA delivery systems for B-cell lymphoma therapy, in which both the aptamer and the Dicer substrate siRNA (DsiRNA) portions are conjugated through a “sticky bridge.” The BAFF-R is overexpressed on the surface of B-cell malignancies, allowing binding and inter...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Design, Assembly, and Evaluation of RNA–Protein Nanostructures
The use of RNA–protein interaction motifs (RNP motifs) to design and build nanoscale objects has the potential to expand the field of RNA nanotechnology. In principle, RNP motifs can be integrated easily into RNA nano objects, providing an alternative technique to increase the functional and structural complexities of the RNA. Investigating the design principles of RNP nanostructures will enable the construction of highly sophisticated biomacromolecular complexes such as ribosomes from scratch. As an initial step towards this goal, we designed and constructed triangular-like nanostructures by employing box C/D kink-t...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Mapping RNA Interactions to Proteins in Virions Using CLIP-Seq
RNA nanotechnology often involves protein–RNA complexes that require significant understanding of how the proteins and RNAs contact each other. The CLIP-Seq (cross-linking immunoprecipitation, and DNA sequencing) protocol can be used to probe the RNA molecules that interact with proteins. We have optimized the procedures for RNA fragmentation, immunoprecipitation, and library construction in CLIP-Seq to map the interactions between the RNA and the capsid of a simple positive-strand RNA virus. The results show that distinct portions of the viral RNA contact the capsid. The protocol should be applicable to other RNA vi...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Multiple Approaches for the Investigation of Bacterial Small Regulatory RNAs Self-assembly
We present here the various approaches that can be used for the detection and analysis of bacterial small noncoding RNA self-assemblies. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Overview of Methods in RNA Nanotechnology: Synthesis, Purification, and Characterization of RNA Nanoparticles
RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149–155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833–842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applicati...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
Mapping Protein–RNA Interactions by RCAP, RNA-Cross-Linking and Peptide Fingerprinting
RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein–RNA contacts within virions. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news
