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A microassay for quantitative determination of β-glucuronidase reporter gene activities in individually selected single higher plant cells
Using computer controlled microtechniques for individually selecting specific single cells and for delivering and maintaining volumes of liquids in the nanoliter range we have developed a procedure to quantitatively assay the beta-glucoronidase (GUS) activity of individual transformed cells. Using standard solutions of the reaction product 4-methylumbelliferone (MU) and determining the appropriate optical parameters for a microscope photometer we established the suitability of the method for quantitative determinations at the single cell level. The enzyme assay was performed after lysis of single cells and subsequent incubation in a total volume of 100 nl lysis buffer, and detection of enzyme activities derived from single cells was easily achieved with 4-methylumbelliferyl glucuronide (MUG) as a substrate. The method was used to address the following problems: (1) variability of GUS-activity among different tobacco mesophyll protoplasts derived from stably transformed plants, (2) determination of the efficiency of different promoters regulating the same reporter gene at the single cell level, (3) the proportion of cells transiently expressing the GUS-gene following PEG treatments and (4) transient expression in individual cells after intranuclear microinjection of appropriate DNA.
beta-glucuronidase microanalysis microinjection nicotiana stable transformation transient expression transformation conditions hybrid genes foreign DNA protoplasts microinjection expression efficiency injection culture fusions
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