Experimental strategies

Gene transfer has been applied in a wide variety of settings for analysis of gene expression. Perhaps the most common use is to identify the functional role of a transfected normal or mutant gene. For this purpose, the coding sequence of the target gene can be cloned into one of numerous commercially developed expression plasmids which provide constitutive or inducible transcription promoters, as well as non-coding sequences necessary for transcription termination and transcript processing events such as capping, polyadenylation, and nuclear-cytoplasmic transport. These properties all contribute to achievement of high level transgene expression in the transfected cell population. Transfected cells are then assessed for changes in specific cellular phenotypes as a result of the introduction and expression of the normal or mutant gene.

Gene transfer has also been used successfully to study the functional attributes of non-coding DNA sequences which are frequently critical in controlling ex pression of specific genes. Evaluation of transcriptional control frequently requires the analysis of regulatoiy nucleotide sequences in gene promoters/enhancers. Likewise, primary transcript processing, nuclear-cytosolic transport, subcellular localization, translational activity, and mRNA stability may be controlled by sequences in intron regions, 5' and/or 3' untranslated regions, and coding regions of a specific transcript. The experimental strategies employed commonly use plasmids in which the sequence of interest is linked with a reporter gene which is placed under regulatoiy control of the test sequence. Thus reporter gene transcription is determined by enhancer/promoter sequences, which generally are not transcribed themselves, while post-transcriptional controls involve sequences located within primary transcripts or mature mRNAs. Manipulation of each category of sequence allows precise identification and characterization of regions which control one or more aspects of the expression pattern of a specific gene. The choice of reporter gene may be influenced by the nature of the aspect of gene expression which is under study as well as the availability of specific assessment methods. Generally speaking, the choice of reporter gene is determined experimentally in each situation and can involve read-out of gene expression via spectrophotometric (e.g. p-galactosidase) (8), fluorescent (e.g. firefly luciferase) (9), or radioactive label (e.g. chloramphenicol acetyltransferase) (10) measurements.

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