基因表达差异显示实验

The chapter will focus on the high level cell-free production of integral membrane proteins having multiple transmembrane segments by using an individual coupled transcription/translation system based on an Escherichia coli S30-extract. We describe in detail the setup and optim ...

Protein synthesis in cell-free systems is an emerging technology already competing with in vivo expression methods. In this chapter the basic principles of continuous-exchange protein synthesizing systems, and protocols for Escherichia coli and wheat germ translation and tran ...

Among the cell-free protein synthesis systems, the wheat germ-based translation system has significant advantages for the high-throughput production of eukaryotic multidomain proteins in folded state. Here, we describe protocols for this cell-free expression system.

The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle a ...

The most popular approach for proteomic analysis is based on the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. Although very effective, the method suffers from a number of limitations, the most serious one being the necessity of expensive and sophistic ...

The availability of the Bacillus anthracis genome sequence allowed for in silico selection of a few hundred open reading frames (ORFs) as putative vaccine candidates. To screen such a vast number of candidate ORFs, without resorting to laborious cloning and protein purification proced ...

Nuclear magnetic resonance (NMR) methods have been developed to determine the three-dimensional structures of proteins, to estimate protein folding, and to discover high-affinity ligands for proteins. However, one of the difficulties encountered in the application of such NMR me ...

In this chapter we describe technical aspects and experimental potential of the two electrodes voltage clamp (TEVC) electrophysiological approach applied to the Xenopus oocyte-expression system. This technique is addressed to the study of a particular class of expressed protei ...

HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for vi ...

The protein truncation test (PTT) is a simple and fast method to screen for biologically relevant gene mutations. The method is based on the size analysis of products resulting from in vitro transcription and translation. Proteins of lower mass than the expected full-length protein represe ...

The single-molecule PCR-linked in vitro expression (SIMPLEX) technology, which can directly link a single molecule of a gene to its encoding protein, has been used to engineer enantioselectivity of lipase from Burkhorderia cepacia KWI-56. A combinatorial mutation has been introduc ...

A large amount of research activity worldwide is currently directed towards developing lipid- or polymer-based, non-viral gene vectors for therapeutic applications. This strong interest is motivated by their low toxicity, ease of production, ability to transfer large pieces of DNA i ...

Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown ...

Mesenchymal stem cells (MSCs) are a promising target for the delivery of secreted proteins due to their ease of isolation, expansion, and genetic modification. The bleeding disorder hemophilia A results from the deficiency of a secreted blood clotting factor termed factor VIII (fVIII). He ...

Noninvasive imaging of molecular-genetic and cellular processes is an effective way to determine the location(s), magnitude, and time variation of action of gene products used for many therapeutic strategies. Lentiviral vectors provide effective means for the delivery, integra ...

Cystic fibrosis (CF) a monogenic lethal disease and, therefore, ideally suited for the development of gene therapy. The first clinical trials were carried out shortly after cloning the CF gene in 1989. Since then, 25 trials have been carried out. Proof of principle for low-level airway gene transf ...

The use of replication-competent oncolytic viruses has largely advanced cancer gene therapy. Oncolytic virus not only possesses unique mechanisms of action that are distinct from other treatment modalities, its self-perpetuating nature provides an ideal platform for therape ...

The development of trans-splicing vectors opens the door for delivering a large therapeutic gene with adeno-associated viral vectors (AAV). One potential application is to deliver the 6 kb mini-dystrophin gene for Duchenne muscular dystrophy (DMD) gene therapy. However, early attem ...

Type 1 diabetes is caused by autoimmune destruction of insulin-producing cells in the pancreas. Type 1 diabetes could potentially be treated by islet transplantation; however, the recurrence of autoimmunity leads to the destruction of islet grafts in a relatively short time frame. Ther ...

Lentiviral vectors are able to transduce non-dividing cells and maintain sustained long-term expression of the transgenes. Many cell types including brain, liver, muscle, and hematopoietic stem cells have been successfully transduced with lentiviral vectors carrying a variety ...