Use Of Reporter Genes Research Articles

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms.

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.

Hybrid genes in the analysis of transformation conditions: II. Transient expression vs stable transformation—analysis of parameters influencing gene expression levels and transformation efficiency

Reports on direct gene transfer have dealt with either the obtention of stable transformants and transgenic plants, or described the use of reporter genes to analyse different aspects of gene expression in plant protoplasts and conditions for their use in transient gene expression assays.In this paper we present comparisons between several transformation techniques, show species‐specific differences in efficiencies of stable transformants and in the levels of transient gene expression, and report on the identification of major parameters responsible for DNA uptake as judged from transient chloramphenicol acetyl transferase (CAT) expression levels and from efficiencies of transformation based on kanamycin‐resistance. The described procedures have been simplified, optimized and standardized and should allow routine use with a great variety of plant species.

82] Use of reporter genes in cyanobacteria

The chapter addresses the utilization of reporter genes and vectors employed in the assessment and detection of transcription regulatory sequences in cyanobacteria. Reporter genes fused to various regulatory sequences have proved useful in analyzing gene expression in many cell systems. They have been used to detect and monitor signals that regulate gene expression. The technique involves the use of a gene trimmed of its promoter and, when desired, of its translating initiation signals. The product of the reporter gene should be readily quantitatable by simple enzymatic assays, and its synthesis should allow scoring or selection of cells expressing the gene. Regulatory elements can be studied by isolating clones expressing the gene, and the level of transcription is determined by assaying its product. Two strategies have been employed for gene cloning in cyanobacteria. One uses shuttle plasmid vectors that carry both a cyanobacterial and an E. coli replicon, each of which functions in its indigenous host. The other strategy exploits the efficient recombination system of Anacystis nidulans R2 and Aphanocapsa PCC 6803 for a directed integration of the cloned gene into the chromosome.