Bench Marks

The generation of transgenic plants can be a lengthy and difficult process. Transient expression assays have been developed as faster and more convenient alternatives for investigating gene function. These assays often take advantage of the ability of Agrobacterium to transfer foreign DNA into plant cells with intact cell walls. Agrobacterium-mediated transformation is, however, inefficient and shows great variability. In the May issue of Cold Spring Harbor Protocols, Andreas Nebenführ and colleagues from the University of Tennessee present FAST Technique for Agrobacterium-Mediated Transient Gene Expression in Seedlings of Arabidopsis and Other Plant Species, a quick, efficient and economical assay for gene function in intact plants. The technique involves cocultivation of young plant seedlings and Agrobacterium in the presence of Silwet-77. The Silwet-77 facilitates transformation, thus replacing a wounding or device-dependent vacuum step. As one of May’s featured articles, it is freely available to subscribers and non-subscribers alike.

Using a promoter that can drive expression at an appropriate level is crucial in designing constructs for gene expression. Promoters can be tested via transient or stable transfection. But transfection efficiency in such assays can be low, so promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein’s activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. The May issue of Cold Spring Harbor Protocols includes updated versions of three commonly used assays for promoter strength.

The Luciferase Assay uses a gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg++, yielding a fluorescent product that can be quantified by measuring the released light with a luminometer. The luciferase assay is extremely rapid, simple, relatively inexpensive, sensitive, and possesses a broad linear range.

The Chloramphenicol Acetyltransferase Assay utilizes an Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene. CAT catalyzes the acetylation of [14C]chloramphenicol which is monitored by autoradiography following thin-layer chromatography (TLC). The percent conversion of [14C]chloramphenicol to acetyl-[14C]chloramphenicol can be measured by PhosphorImager analysis of the TLC plate, counting in a scintillation counter, or by densitometry analysis of an autoradiograph.

The Beta-Galactosidase Assay uses the E. coli lacZ gene which encodes a beta-galactosidase. Beta-gal activity is measured through a simple and inexpensive colorimetric assay. Cells are lysed and extracts are mixed with O-nitrophenyl-beta-D-galactopyranoside (ONPG), which results in a yellow product. The optical densities of the samples are then determined spectrophotometrically.

The large size and external development of the frog Xenopus laevis make it an ideal system for in vivo imaging of dynamic cellular activity. Xenopus embryos are amenable to simple genetic manipulation techniques including knockdowns and misexpression, as well as transgenesis. The ease of collecting large numbers of embryos and the larger size of individual cells within an embryo as compared with other vertebrate model systems provides an excellent platform for the observation of cellular behavior and subcellular processes. In the May issue of Cold Spring Harbor Protocols, John Wallingford and colleagues from the University of Texas provide a suite of articles detailing live imaging of Xenopus laevis at low magnification, confocal imaging of fixed tissues, and in one of May’s featured articles, High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology. This protocol describes methods for labeling and high-magnification time-lapse imaging by confocal microscopy. Like all of our featured articles, it’s freely available to subscribers and non-subscribers alike.