细胞技术

RNA interference (RNAi) libraries have been proven to be a powerful tool for large-scale functional genetic screens. To facilitate high-throughput functional genetic screens in embryonic stem cells, a system for construction of random dsRNA expressing RNAi libraries was develop ...

Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive ge ...

Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNAi-based screens in primary human hematopoietic stem and progenitor cells. These cells are suitable ta ...

Murine embryonic stem cells (ES) are pluripotent cells and have the potential to become a wide variety of specialized cell types. Mouse ES cell differentiation can be regarded as a valuable biological tool that has led to major advances in our understanding of cell and developmental biology. In v ...

Recently, RNAi, including microRNAs (miRNAs), has become an important tool to investigate the regulatory mechanism of stem cell maintenance and differentiation. In this short chapter, we will give a brief overview of the discovery history, functions, and mechanisms of RNAi and miRNAs. We w ...

RNAi is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-strand RNA molecules. In the 11 years since the initial report, RNAi has now been demonstrated to function in mammalian cells to alter gene expression and used as a means for genetic discovery as well as a possib ...

Glioblastoma multiforme (GBM) are the most common malignant brain tumours in adults, characterized by short survival periods of patients. Their aggressive local growth pattern and increased invasiveness, due to a high motility of the tumour cells, hamper treatment. However, the mole ...

Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and ren ...

Single-cell manipulation supporting robot (SMSR) has enabled femtoinjection, a high-throughput and semi-quantitative microinjection in the range of femtogram (fg) DNA and other molecules. An enhanced green fluorescent protein (EGFP) gene expression vector can be introduced ...

Osteoclasts and their precursors have traditionally been considered difficult cells to transfect using standard approaches. Here, we describe several methods for transfection of mature osteoclasts and their precursors using the Amaxa™ Nucleofector system, lentiviruses, ...

Transcription is the process by which the rate of RNA synthesis is regulated. Here, we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays, three commonly used methods for studying g ...

This chapter describes the analysis of signalling pathways in bone cells by the use of western blotting and immunoprecipitation, including a step by step guide to cell culture techniques, protein isolation, purification, measurement, electrophoretic transfer, and detection.

Here, we present methods for extracting DNA and RNA from samples of whole bone tissue and culture bone cells and describe methods quantitative and qualitative measurement of the extracted nucleic acids. These protocols described provide high-quality nucleic acids suitable for down ...

Bone histomorphometry remains an important tool to study the pathophysiology of bone disease and the cellular mechanism by which treatments work. Here, we review the methods for embedding, sectioning, staining, and analysis of bone sections in rodents.

This chapter describes methods for quantitation of gene expression in bone cells and bone tissues using the technique of quantitative reverse transcription-polymerase chain reaction.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with ...

Here, we described a method for carrying out nonradioactive in situ hybridization to detect mRNA transcripts in cryostat sections of mouse bone using the CryoJane� Tape-Transfer System and digoxigenin (DIG)-labeled riboprobes.

There has been great interest in the identifying the mechanisms by which apoptosis is regulated in bone over recent years and in the biological role that this process plays in bone metabolism and bone disease. Here, we describe several methods for the detection of apoptosis in bone sections and in bo ...

This chapter described methods for Scanning Electron Microscopical imaging of bone and bone cells. Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. This chapter co ...

This chapter describes procedures to process mineralized tissues obtained from different sources for transmission electron microscopy (TEM). Methods for fixation, resin embedding, staining of semi-thin sections and ultrathin sections are presented. In addition, attenti ...