遗传学实验技术

Over the past few years protein transduction has emerged as a powerful means for the delivery of proteins into cultured cells and into whole mice. This method is based on the ability of proteins containing protein transduction domains (PTDs), short stretches of 9–16 predominantly basic amino a ...

A two-step cloning system for expression of foreign and native genes from heterologous promoters in single copy from the E. coli chromosome is described. The system is based on the conditional-replication integration and modular CRIM plasmid technology and new CRIM plasmids described h ...

Conversion of some carbon sources into desired compounds by a biological system is the goal of many biotechnologists. The understanding of the mechanisms by which an organism does these conversions permits the improved production of specific metabolites. Pathway engineering invo ...

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insert ...

Exchange of the promoter of a gene in the genome for another promoter whose expression can be controlled easily can overcome problems associated with the expression of the same gene from a promoter on a plasmid. Some genes are difficult or impossible to clone in plasmid-based vectors and often a stab ...

The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. To serve the increasing demand for clones expressing different cDNAs, we developed a cultivation and induction protocol amenable to automation to increase the number of c ...

We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducib ...

Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette con ...

Filamentous fungi include a large, heterogeneous group of heterotrophic organisms with profound influence in human activities. In spite of their economic and scientific relevance, little information is available at the molecular level about their biology. The development of gen ...

Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research. In addition, they are excellent models for molecular and genetic studies. An important key in the advancement of geneti ...

Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders—that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to kno ...

Plant-based expression of recombinant proteins offers significant advantages over transgenic animal-and cell-based systems. Unlike bacteria, plants perform the complex protein-processing steps required to produce eukaryotic proteins in active form. In order to facili ...

The chloroplast genome is ideal for engineering because it offers a number of attractive advantages, including high-level gene expression, the feasibility of expressing multiple genes or pathways in a single transformation event, and transgene containment due to lack of pollen tran ...

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes t ...

Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them (1). In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional ...

Manipulation of the eukaryotic genome not only has contributed to the progress in our knowledge of multicellular organisms but has also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility. There are new and va ...

Chromosomal position effects are the cause of variegation of transgene expression relating to the eukaryotic genome environment. These effects modify heterologous transgene expression, presumably due to dominant epigenetic marks that cause variegated or gradual silenci ...

Inducible expression of desired transgenes in mammalian cells and animals is a current priority in basic and applied research, biopharmaceutical manufacturing, gene therapy, and tissue engineering, as well as in drug discovery. Among the most prominent human-compatible transge ...

The creation of recombinant mammalian cells with defined expression levels requires extensive screening. This is mainly due to the unpredictable site and copy number of the recombinant DNA in the hosts’ chromosomal DNA. The method presented here is based on the exchange of expression cass ...

The purpose of this chapter is to propose a practical procedure for the generation and selection of Chinese hamster ovary cells producing high levels of recombinant protein by combining in vitro and in vivo amplification of the foreign gene. A detailed description of the expression and ampli ...