遗传学实验技术

Gene delivery methods play a key role in molecular neurobiology. The possibility to efficiently deliver and express genes not only in cell lines, but also in neurons in culture in vivo has facilitated studies on the understanding of gene function. Generally, neurons are not very susceptible to g ...

Recombinant protein expression is one of the basic elements of modern molecular biology. The completion of the sequencing of the human genome has, in fact, accelerated the need of rapid and efficient expression systems. Extensive efforts have been dedicated to the development of expressi ...

Retroviral vectors developed from the murine leukemia virus (MLV) were the first and most widely used vectors for gene transfer experiments (1). Delineation of their replication strategy allowed extensive manipulation of their genome and the development of vector packaging cell li ...

Retroviral vectors have become important tools in gene therapy as a result of a number of highly desirable properties, including stable genomic integration, which are rarely found with other gene delivery vectors. However, after two decades of experience with this technology, some shor ...

This chapter aims to describe the main procedures involved in constructing a disabled recombinant herpes simplex virus type 1 (HSV-1) vector. However, before describing the methodology in detail, it is important to briefly consider the structure and life cycle of HSV1 and the modificatio ...

Herpes simplex virus type 1 (HSV-1) has several unique features that make it a promising candidate for gene transfer vehicles. These include: i) a pantropic viral envelope; ii) a large genome (approx 152 kb), which confers its large transgene capacity; and iii) the ability of entering a latency state in ...

The key fundamental problem for effective gene therapy remains gene delivery. Gene therapy has achieved some modest success in animal and human experiments (1–7). However, effective gene delivery remains the chief obstacle. Herpes simplex virus type 1 (HSV-1) vectors offer several impr ...

One of the challenges of current gene therapy vector development, concerns targeting a therapeutic gene to diseased cells with the aim of achieving sufficient gene expression in the affected tissue, while minimizing toxicity and expression in other tissues. The use of recombinant aden ...

Adenovirus (Ad) vectors have been used to successfully deliver genes into a wide variety of non-central nervous system (CNS) tissues and organs in animal models of human disease and in several human phase I clinical trials (1–3). Adenoviruses are easily purified to the high titers required for in v ...

Adenovirus (Ad) vector-mediated gene transfer is useful in applications where transient, high-level transgene expression is required. In contrast to onco-retroviral vectors, Ad vectors efficiently infect nondividing cells. Most recombinant Ad vectors currently used for in v ...

Gene transfer using adenovirus (Ad) can be a highly efficient method for transducing cells and tissues. Wild-type Ad has a 36-kb double-stranded linear genome that encodes numerous overlapping open reading frames (1). Because wild-type Ad is infectious to humans, vectors used in the labora ...

The effectiveness of gene therapy is essentially governed by the ability of the vector to reach the relevant tissue, and once there, to facilitate the expression of appropriate quantities of the gene product. Over the course of evolution, viruses have developed highly specialized and succe ...

Calcium phosphate mediated transfection of genomic DNA samples into mammalian recipient cells can be used to isolate specific genes of interest. In theory, for any phenotype for which there is a suitable selection in cell culture, a gene sequence encoding that phenotype can be transferred a ...

Evaluation of single-locus probe results in criminal-identity cases is best presented within the standard forensic framework for handling trace evidence. A comprehensive account was given by Aitken (1), and the language of that text will be used here. For simplicity, suppose a stain from the ...

Since its discovery by Prof. Sir Alec Jeffreys FRS in 1984, DNA fingerprinting using multilocus VNTR probes (MLPs) has been widely used in the field of human identification (1,2). Although multilocus probes were used in forensic investigations in the late 1980s (3), forensic samples often yield ...

PCR-VNTRs are important markers for questions of identification, individualization, and discrimination. They already form an integral part of forensic DNA analysis. They can be subdivided into the systems of short tandem repeats (STR) with fragment lengths ranging between approx 1 ...

Highly polymorphic variable number tandem repeat (VNTR) loci have proven very useful for human DNA testing purposes (1-3). Initially, these variants were characterized by restriction fragment-length polymorphism (RFLP) analysis. Subsequently, the polymerase chain reacti ...

PCR optimization is usually performed in order to obtain maximum specificity and yield. In some applications for which the amount of template may be limiting, or when there is a large amount of nontarget sequences, the sensitivity is maximized. Nonoptimized conditions promote artifactu ...

Short tandem repeat (STR) loci consist of repetitive elements of 3-7 nucleotides. The STR loci, which are numerous in the human genome, are highly polymorphic in length and may also vary in the sequences of the repetitive elements. The polymerase chain reaction (PCR) makes it possible to analyze ve ...

This introduction is intended to inform the forensic scientist of the historical development of STR typing on automated sequencers, as these precedents will have a bearing on the techniques and equipment used in the present-day laboratory. The section then concludes with a brief outline of ...