Methods in molecular biology
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Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998), human embryonic stem cells (hESCs) have been derived from blastocysts, and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs, fibroblast feeder layers are often used at some phase in the culturing protocol. ⋯ These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose.
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Duchenne muscular dystrophy (DMD) is a severe muscle wasting X-linked genetic disease caused by dystrophin gene mutations. Gene replacement therapy aims to transfer a functional full-length dystrophin cDNA or a quasi micro/mini-gene into the muscle. ⋯ Further modification/optimization of these microgene vectors may improve the therapeutic potency. In this chapter, we describe a species-specific, codon optimization protocol to improve microdystrophin gene therapy in the mdx model.
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The posttranslational modification of proteins is important for the regulation of enzymatic activity, protein half-life, and interaction with other molecules. One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. ⋯ Furthermore, phosphoproteome analyses are incompatible with long organelle isolation procedures prior to analysis, because of the highly dynamic nature of regulatory phosphorylations. In this chapter, we provide a detailed step-by-step overview of the complex experimental setup required for successful chloroplast phosphoproteome analysis, report our experience with existing methods, and comment on their application in the field.
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Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.
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Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. ⋯ Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosphorylation sites and profile their relative abundance during differentiation. This systems-biology-based approach provides new insights into how human pluripotent stem cells exit the pluripotent state.