遗传学实验技术

Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is especially simple, inexpensive, and safe. However, applications of this method have been limited by the relatively low expression levels of the transferred gene. Recently, we invest ...

With the aim of developing successful human gene therapy, numerous viral and nonviral (synthetic) methods of gene transfer have been developed (1,2), each method having limitations as well as advantages. To develop in vivo gene transfer vectors with high efficiency and low toxicity, sever ...

For in vivo gene transfer, synthetic (nonviral) vectors are thought to have several advantages compared with viral vectors: they can accommodate largesize DNA molecules (therapeutic genes including their endogenous regulatory regions), be used under reduced confinement cond ...

Liposome-Polycation-DNA (LPD) nanoparticles is a novel nonviral vector developed in our laboratory for efficient systemic gene delivery. Currently there are two LPD formulations in use, differing in cationic liposome composition. One is composed of cationic lipid dioleoyl-tr ...

Site-specific correction of single-base mutations in target DNA sequences has been developed using an RNA-DNA hybrid oligonucleotide (RDO) to correct or cause a specific point mutation in episomal and genomic DNA (1–14). In its original design, an RDO was composed of two strands, one strand co ...

Following direct intramuscular injection of plasmids, transgene expression follows a biphasic pattern in which expression levels are maintained for approximately 1 month, and then decline to trace levels that persist for 1 year or more (1,2). However, even during the first month, transg ...

The direct cloning of DNA fragments, either derived from naturally occurring or artificially designed gene sequences, into various cloning vectors, including bacteriophages, plasmids, and viruses, is the cornerstone of modern molecular biology. Such recombinant constructs ...

Expression of proteins from cDNA clones by transcription and translation in vitro can be an attractive option for analytical applications that do not require significant quantities of product. Our laboratory has been interested in this technique because the translation of nucleic a ...

The production of proteins is one of the main applications of genetic engineering in biotechnology. Even though standard cloning procedures are now routine and a large variety of host-vector systems for gene expression are available, difficulties are encountered when theoretical s ...

Since introduction of the first pUC plasmids (1), a great variety of plasmid vectors that use α-complementation and expression from the lac promoter, or its derivatives tac and trc promoters, have been developed (e.g., see refs. 2–14). In order to maximize utilization of these vectors, various Esc ...

Bacteriophage λ has been in use as a cloning vector for over 20 years, and has been extensively used as an expression vector. A historical overview of λ as a cloning vector can be found in Murray (1), and a more recent review of λ vectors is in Chauthaiwale et al. (2). In general, λ is more useful as a tool for expressing foreign ...

Recombinant DNA technology enabling genes or gene fragments to be spliced to form fusions has become an important objective in many fields of research. The fusion proteins, expressed from such operatively linked genes, often display the combined properties of the parent proteins and are u ...

The promoter of the E. coli tryptophan (trp) operon has proven to be a workhorse for the production of hundreds of proteins; from small scale to pharmaceutical production levels (1). The trp promoter is strong, easily regulated, and well characterized. Transcription of a cloned gene from a trp promo ...

Most commonly used prokaryotic expression vectors fall into one of the following three categories: 1. They have a low copy number and a low yield of single-stranded DNA, 2. They have a high copy number and the

Expression systems for the production of heterologous proteins are now available in many different host organisms such as Escherichia coli, yeast, baculovirus and mammalian cells. The choice of an appropriate host organism is dictated by the particular protein to be expressed, and by req ...

In the past decade we have witnessed rapid advances in the development of systems for expression of recombinant proteins. A wide range of expression systems, host organisms, and processing procedures have been described. Among these, E. coli remains an important organism for production of r ...

The yeast Saccharomyces cerevisiae provides an excellent system to study genes of eukaryotes because it has been extensively characterized genetically and because the molecular mechanisms governing many cellular processes in yeasts are conserved in other organisms. For exam ...

The yeast Saccharomyces cerevisiae can be grown in the laboratory with ease and at relatively low expense, and can be propagated in large scale fermentation cultures when preparation of larger amounts of a recombinant protein is desired. The S. cerevisiae genome can be manipulated to reduce p ...

The promoter of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) has been used to construct vectors for expression of heterologous proteins in budding yeast (Saccharomyces cerevisiae) (1–4). This promoter is one of the most efficient yeast promoters and is us ...

The ADH2 promoter belongs to a class of promoters subject to catabolite repression. Many of these promoters have strong transcriptional start signals, which are repressed by the presence of glucose. The strength of the ADH2 promoter coupled with the fact that the promoter is tightly regulat ...