遗传学实验技术

Since its development in 1983 (1), Ribonuclease Protection Assay (RPA) has become a widely employed technique for RNA analysis. The procedure is based on hybridization of the RNA being analyzed to a labeled (nonisotopic or radioactive) antisense RNA probe with successive digestion of unh ...

The ability to detect Shigella in foods is often hampered by the often-low numbers of organisms present in the sample at the time of analysis. At present, a reliable, time-efficient, and highly sensitive protocol to isolate Shigella from foods is unavailable although a conventional method is cu ...

There are several species of Listeria, but only Listeria monocytogenes has been identified as the principal pathogen in humans and animals. L. monocytogenes is a ubiquitous Gram-positive bacterium responsible for an uncommon but potentially serious infection in humans who ingest co ...

Biotin is a small vitamin found in tissue and blood and is synthesized by intestinal bacteria. Biotin functions as a prosthetic group for several carboxylases and as a CO2 carrier. The molecule consists of a ring system covalently linked to the enzyme by a valerate side chain acting as a flexible arm. Av ...

Nick translation was the first method devised for the in vitro labeling of DNA (1). During the reaction the DNA to be labeled is nicked by DNase I yielding a free 3′ hydroxyl end. DNA polymerase I then adds a new nucleotide to this end. The 5′–3′ exonuclease activity of the polymerase then moves the “nick” along the st ...

Fluorescence in situ hybridization (FISH) of whole cells using 16S rRNA-targeted oligonucleotide probes is a powerful technique with which to evaluate the phylogenetic identity, morphology, number, and spatial arrangements of microorganisms in environmental settings (1). P ...

Labeling of DNA by nick translation has three major drawbacks: the time taken to perform the reaction (at least 1 h), the temperature sensitivity of the reaction, and the low specific activity of the probes generated. Random primed labeling developed by Feinberg and Vogelstein (1,2) solves all of t ...

The progression through the cell cycle is monitored by positive and negative regulators. One family of negative regulators has been reported to act as cyclin-dependent kinase inhibitors (CKI) (1–3); and these, in turn, have been subdivided into two groups on the basis of sequence homology. The f ...

Leishmania is a protozoan parasite belonging to the order Kinetoplastida, family Trypanosomatidae, and genus Leishmania. These parasites are the causative agents of the disease known as leishmaniasis, which in humans has a broad spectrum of clinical manifestations depending on the ...

Multiple myeloma is a hematological malignancy characterized by an increased number of bone marrow plasma cells and the presence of high levels of a serum monoclonal immunoglobulin that is coded by a unique genetic sequence in the variable region of the immunoglobulin heavy chain gene. Mor ...

The identification of specific viral and tumor antigen epitopes recognized by CD4+ or CD8+ T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope ...

Monitoring the immune response is an essential aspect of numerous clinical vaccination trials in order to evaluate the efficacy. In these clinical vaccination trials, peripheral blood mononuclear cells (PBMC) are isolated at different time points from patient blood samples and sub ...

The observation that dendritic cells (DCs) charged with tumor-associated antigens (TAAs) is a potent strategy to elicit protective immunity in tumor-bearings hosts has prompted extensive testing of DCs as cellular adjuvant in cancer vaccines. To improve the clinical development of ...

mRNA-based vaccines are currently being developed for treating various diseases including cancers. For this purpose, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen offers several advantages over plasmid DNA encoding tumor antigen including better delive ...

Synthetic antigen-encoding mRNA is increasingly exploited as a tool for delivery of genetic information of complete antigens into professional antigen presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. Two strateg ...

Changing cell fate without altering its genome is very desirable in many experimental systems and for cell therapy. Compared to DNA plasmid or viral-based approach, mRNA has the advantage of high transfection efficiency, no danger of changing the genome or creating mutational insertion ...

Natural killer (NK) cells are emerging as a new tool for cell therapy of cancer. However, some cancer subtypes are relatively resistant to NK cell cytotoxicity. Expression of anti-CD19 chimeric signaling receptors can enhance NK-cell reactivity against CD19+ leukemia and lymphoma cells. ...

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to ...

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electro ...

Transfecting with in vitro transcribed, protein-encoding mRNA is a simple yet effective method to express high levels of the desired RNA-encoded proteins in primary cells. Cells can be transfected with antigen-encoding mRNA, which is translated into protein and is processed by the cellu ...