实验基础

Since the polymerase chain reaction (PCR) technique was developed in 1986 (1), it has found wide application in molecular biology and is now regarded as an irreplaceable tool in many laboratories. This chapter will deal with its use for the detection of virus genomes for diagnostic and experimen ...

The transfer of DNA sequences into a population of cells can rarely, if ever, be achieved with 100% efficiency. Typically, transfection of cells with the calcium phosphate method will transduce only between 0.1 and 1% of the cells with the sequences of interest (1), although some workers have achiev ...

When using recombinant retroviruses for gene transduction, it is necessary to have a measurement of the concentration of virus particles in the medium conditioned by virus-producing cells (i.e., the viral titer). In the case of viruses encoding selectable markers, this can readily be deter ...

This chapter aims to provide the reader with the experimental protocols required to produce a baculovirus containing the gene of interest, including transfection of Spodoptera frugiperda cells and the screening of progeny virus by plaque assay. In addition, an outline of the biochemic ...

Retroviral vectors are uniquely suitable for high-efficiency gene transfer to a large target cell population and this (among other things) has led to the exploration of previously impractical strategies for somatic gene therapy (1,2). Thus, for example, cultured bone marrow can be used to r ...

Baculoviruses have been used for many years as effective pest-control agents; however, their interest to molecular virologists stems from their exploitation as helper-independent viral expression vectors for the production of proteins in a eukaryotic environment. The pionee ...

The self-assembly properties of a protein encoded by the TYA gene of the yeast Ty element can be exploited to produce hybrid Ty-VLPs (virus-like particles) (1,2). There has been developed a series of expression vectors that allow the construction of Ty fusion genes containing protein coding seq ...

The synthesis of recombinant proteins or protein domains in microbial, insect, or mammalian systems is now commonplace in molecular biology laboratories. The gene or gene fragment encoding the protein of interest is inserted into a specialized expression vector, flanked by efficient ...

Chapter 22 outlines the construction of engineered, full-length poliovirus cDNAs in which the region encoding a well-characterized antigenic site has been replaced by sequences of choice. This chapter briefly describes the methods used to generate, maintain, and characterize inf ...

We have developed the very safe and efficacious live-attenuated Sabin 1 poliovirus vaccine strain as a vehicle for the presentation of defined epitopes from foreign pathogens (1–3). Precise modification of the poliovirus capsid is made possible by the application of recombinant DNA tec ...

In Chapter 20, a general procedure was described for the construction of the intermediate vectors necessary for the insertion of foreign DNA sequences into the vaccinia virus (Vv) genome. The principles and basic methodology for the isolation of recombinant vaccinia viruses will be disc ...

Vaccinia virus (Vv) is a member of the genus Orthopoxvirus, one of seven genera included in the family Poxviridae. Most of these viruses infect vertebrates (Orthopoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Parapoxvirus), but one genus, Entomopoxvir ...

Epstein-Barr virus (EBV) is able to immortalize human B-lymphocytes with high efficiency. This property underlies the role of EBV in a number of human diseases. First, EBV is the causative agent of infectious mononucleosis, which is a benign proliferation of B-lymphocytes (1). Second, EBV is in ...

Vectors containing elements of the Epstein-Barr virus (EBV) genome are primarily used to maintain cloned DNA inserts as plasmids in mammalian cells. In addition, EBV-based vectors are proving to be valuable tools in elucidating details of EBV biology that have long eluded students of the vir ...

In order to use herpes simplex virus (HSV) as a vector for the transmission and expression of foreign genes, it is obviously necessary to be able to prepare stocks of the virus and to propagate both HSV and the recombinant viruses derived from it. Similarly, the introduction of foreign genes into the virus ...

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) are doublestranded DNA viruses with a genome size of 152 kbp. The genome consists of two unique regions, UL (long) and Us (short), flanked by repeated sequences (Fig. 1; for review, see ref. 1). The two viruses are closely related, and both infect humans, produ ...

Most of the time, retrovirus vectors retain only cis-acting sequences from the original viral genome. These sequences allow the recombinant structure to be transcribed (LTR promoter/enhancer) and the RNA to be processed (splicing and polyadenylation signals), packaged into a virion ...

Human papillomavirus (HPV) research dates back to the turn of the century, when Ciuffo demonstrated that human warts are caused by an infectious agent, only later identified as a virus (1). More recently, the HPVs have generated particular interest, since they include some of the few viruses clea ...

COS-1 cells were created by transforming an established line of monkey epithelial cells, CV-1, with a defective mutant of SV40 (1). The SV40 mutant used carried a small deletion within the origin of replication and, although this construct transformed CV-1 cells, which are permissive for lytic g ...

On account of their relatively small, defined genomes, viruses have long been a convenient model system in which to study eucaryotic gene expression. Usually, viral infection is followed by early gene expression, which allows the subsequent processes of viral DNA replication and late gene ...