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Analysis of X-chromosome inactivation patterns can be a useful tool in the identification of carriers of certain X-linked diseases and also for other investigations, such as gene mapping and clonality analysis. X inactivation is the process in females whereby one of the two X chromosomes of a ce ...

Every genetic laboratory diagnosis carries some degree of uncertainty, even if only the risk of laboratory error. Increasingly, diagnosis is based on direct tests that tell us whether or not the patient carries a given mutation. In these cases formal risk analysis is scarcely necessary—alt ...

The hemoglobinopathies are a diverse group of inherited recessive disorders consisting of the structural hemoglobin variants and the thalassemias. They can occur at very high carrier frequencies in the malarious regions of the world and are regionally specific, with each population ...

The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. This ...

Mechanical loading of chondrocytes in 3D constructs has been used to investigate mechanotransduction and its potential for stimulating tissue-engineered cartilage repair. This chapter describes the preparation of 3D agarose or alginate constructs seeded with isolated cho ...

The mitogen-activated protein (MAP) kinase family consists of extracellular signal-regulated protein kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) and transduces signals from the extracellular environment to the cytoplasm and nucleus. MAP kinase signaling invol ...

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered c ...

A classic method for the encapsulation and culture of chondrocytes in alginate beads is described. Chondrocytes are released from cartilage matrix by collagenase/dispase digestion and mixed with a solution of 1.25% alginic acid until a homogenous suspension is obtained. The suspens ...

The adult hematopoietic system is repopulated in its entirety from a rare cell type known as hematopoietic stem cells (HSCs) that reside in the marrow space throughout the skeletal system. Here we describe the isolation and identification of HSCs both phenotypically and functionally.

Osteoclasts are the only cell type in the body to resorb bone. Osteoclasts play a critical role in physiologic and pathologic bone remodeling. Many genetic mouse models affect the skeleton by regulating osteoclast function directly or indirectly. This protocol describes a procedure for ...

This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a coll ...

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived “mesenchymal stem cells”) contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). The biological properties of BMSC cultures are assessed by a va ...

To identify factors that are necessary and sufficient for chondrocyte hypertrophic differentiation and cartilage matrix mineralization, primary chondrocyte culture models have been developed. Here we describe the isolation, short-term and long-term culture, and analysis ...

High-density micromass cultures of embryonic mesenchymal cells have proved to be an invaluable model for studying the entire chondrogenic program, from precartilaginous condensations through to chondrocyte hypertrophy. This culture model also provides a powerful system in ...

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major meth ...

At least two distinct modalities of cell death, apoptosis and necrosis, can be distinguished based on differences in morphological, biochemical, and molecular changes of dying cells . Cell death is involved both in physiological and pathological conditions of the skeleton: for example, ...

Assessing cell proliferation in situ is an important phenotyping component of skeletal tissues from development to adult stages and disease. Various methods exist including immunostaining for proteins and protein modifications associated with specific steps of the cell cycle, ...

In situ hybridization (ISH) using RNA probes is a valuable technique to characterize gene expression patterns in animal tissues. It provides valuable spatial information about gene expression. Compared to the nonradioactive alternatives,35S radioactive ISH generally provi ...

Spatial and temporal visualization of RNA transcripts in tissue is a key tool in studying both developmental and pathological processes. In situ hybridization is a highly sensitive method for RNA transcript detection. It is based on sequence complementation between a labeled RNA probe a ...

Whole-mount in situ hybridization (WISH) is a highly sensitive method for spatial and temporal visualization of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying both embryonic development and disease. It is based on sequence complementation b ...