Detection Of Gene Expression

Thought should also be given to choosing the most sensitive detection method for every application of nonviral delivery rather than using the method that seems most simple. For example, detection of p-galactosidase expression is far more sensitive than that for the green fluorescent protein (GFP). Specifically, 500 molecules of p-galactosidase (p-gal) per cell are required for detection using X-gal staining, whereas about 1 million molecules of GFP per cell are required for direct detection. Furthermore, detection of GFP may be impossible if the fluorescence background of the target cell or tissue is too high. Detection of chloramphenicol acetyltransferase (CAT) is extremely sensitive with little or no background detected in untransfected cells. Often, assays for CAT expression can provide more useful information than using p-gal or GFP as reporter genes.

Few molecules of luciferase in a cell can be detected by luminescence assays of cell or tissue extracts posttransfection. The sensitivity of these assays is highly dependent on the type of instrument used to measure luminescence. However, luciferase results may not predict the therapeutic potential of a nonviral delivery system. For example, if several hundred or thousand molecules per cell of a therapeutic gene are required to produce efficacy for a certain disease, then production of only few molecules will not be adequate. If only few molecules of luciferase are produced in the target cell using a specific nonviral delivery system, then the investigator may be misled in using this system for therapeutic applications.

Furthermore, noninvasive detection of luciferase expression in vivo is not as sensitive as luminescence assays of cell or tissue extracts posttransfection. Recently, my colleagues tried cooled charge-coupled device (CCD) camera imaging on live mice after intravenous injection of other cationic liposomes complexed to plasmid DNA-encoding luciferase, and they were not able to detect any transfection. However, these liposomal delivery systems had been used to detect luciferase by luminescence assays of organ extracts. My colleagues detected luciferase expression by CCD imaging after intravenous injections of BIV DOTAP:Chol-luciferase DNA-liposome complexes (44). Because the luciferase protein is short-lived, maximal expression was detected at 5 h posttransfection, whereas detection of herpes simplex virus-thymidine kinase (HSV-TK) gene expression using microPET imaging in the same mice was highest at 24 h posttransfection. In contrast to luciferase, the CAT protein accumulates over time, and therefore, the investigator is not restricted to a narrow time frame for assaying gene expression. Furthermore, detection of CAT seems to be more sensitive than CCD imaging of luciferase following intravenous injections of DNA-liposome complexes. However, the animals must be sacrificed in order to perform CAT assays on tissue or organ extracts. In summary, further work is still needed to develop in vivo detection systems that have high sensitivity and low background.

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