细胞技术

Expansion of a homomeric stretch of glutamine residues beyond a critical threshold can produce neurodegenerative disease. This observation led to the idea that abnormal polyglutamine stretches can alter protein structure in ways that contribute to disease. Because they are prone to ...

Antibodies can be extremely useful tools for the field of triplet repeat diseases. These reagents are important for localizing proteins in tissues, and within cells, they can be used in the isolation and characterization of the components of protein complexes, they can distinguish protei ...

Myotonic dystrophy (DM1) is a neuromuscular disorder caused by a CTGn expansion in the 3′-untranslated region (UTR) of myotonic dystrophy protein kinase (DMPK). SIX5 is a homeodomain gene located just downstream of the repeat, and myotonic dystrophy WD protein (DMWD) is located close upstr ...

Small-pool polymerase chain reaction (PCR) constitutes the PCR amplification of a trinucleotide repeat in multiple small pools of input DNA containing in the order of from 0.5 to 200 genome equivalents. Products are resolved by agarose gel electrophoresis and detected by Southern blot hy ...

To facilitate identification of disease genes containing an expanded trinucleotide repeat, a repeat expansion detection (RED) and gene cloning system was established. The RED method was developed to enable detection of expanded trinucleotide repeat sequences in any DNA sample fr ...

The unusual genetic features of trinucleotide repeat (TNR) diseases have stimulated a substantial body of research into the underlying molecular mechanisms of repeat instability. As one useful tool to study TNR instability, selectable genetic assays for expansions and contract ...

Expansions of triplet repeats are responsible for more than 15 hereditary neurological disorders in humans (1,2). Triplet repeats are fairly stable when the number of elementary units is under approx 30, but become polymorphic in length with a clear bias for expansions when this threshold is e ...

Since their discovery in 1991, triplet repeat mutations have been found to be the cause of genomic fragile sites, two of which are linked to mental retardation, myotonic dystrophy, and several late-onset neurodegenerative diseases. In all cases, these mutations exhibit gametic and/or som ...

B-lymphopoiesis in vivo is a very complex process that is influenced by cooperation between cells, cytokines, and other receptor-ligand interactions, which developmentally occur at different cellular stages. Various in vitro models have been very useful in unraveling this complex ...

A chicken DT40 B-cell line provides an excellent model system for the analysis of the function of genes related to B-cell antigen receptor (BCR) signaling. Crosslinking of BCR by �-chain-specific antibody stimulates DT40 cells to undergo apoptosis. Activation of protein-tyrosine kina ...

Over the past 15 yr, the use of transgenic mice has led to significant advances in our understanding of immunological tolerance. In a normal repertoire the number of B cells with a single antigen receptor specificity is very small, making the study of their fate difficult. In contrast, animals that ca ...

Recent advances in cell biology have provided evidence that the plasma membrane is not a homogeneous lipid bilayer but rather contains within it sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, which serve as platforms for both receptor signaling and tr ...

Signal transduction by the B-cell antigen receptor (BCR) regulates development, survival, and clonal expansion of B cells. The BCR complex comprises the membrane-bound immunoglobulin molecule and the Ig-α/Ig-β heterodimer, and was shown to form oligomeric structures. Antigen-me ...

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-t ...

The development and functional activities of B lymphocytes critically depend on their migratory capacity. Both in vitro and in vivo assays can be used to assess the migratory ability of B cells. Filter-based transwell assays measure both spontaneous and chemoattractant-induced cell m ...

During an immune response, B lymphocytes can switch expression of immunoglobulin (Ig) class (isotype) from IgM to IgG, IgE, or IgA. This Ig class switch is based on a deoxyribonucleic acid (DNA) recombination event that results in an exchange of the gene segments coding for the constant region of the Ig ...

Primary murine splenic B cells can be cultured ex vivo and, following treatment with LPS, cytokines and/or CD40L proliferate, and undergo class switch recombination and terminal differentiation to become immunoglobulin-secreting plasmacytic cells. Methods are described here ...

To determine whether an immune system is able to support T-cell-dependent affinity maturation one needs antigens that induce well-characterized immune responses. This chapter describes the response of the BALB/c mouse to hapten 2-phenyl-4-ethoxymethylene-oxazolone (phOx) c ...

This chapter describes the analysis of long- and short-lived plasma cells on tissue sections. Mice are immunized with 4-hydroxy-3-nitrophenyl acetyl (NP) coupled to a T-cell-dependent carrier. Antigen-specific germinal center cells and extrafollicular plasma cells are identi ...

Since its first application in mice almost 10 yr ago, the Cre/loxP has become the system of choice to study gene function in vivo in a cell-type, stage-specific, and inducible manner. This chapter provides a set of updated protocols that will help the reader to construct a vector for conditional gene tar ...