As most graduate students will tell you, cloning can be a time-consuming, difficult, and frustrating experience. This is especially true when the cloning vector is large or lacking many unique restriction sites. The Drosophila vectors pCaSpeR and pUAST, commonly used to generate transgenic flies, are both larger than 8 kb and have a small number of unique restriction enzyme cloning sites. In this issue, Le et al. have tried to improve this situation by creating the next generation of pCaSpeR and pUAST vectors possessing larger numbers of unique cloning sites and by adding matching, smaller shuttle vectors to facilitate modifications, such as site-directed mutagenesis, prior to cloning into the larger transformation vectors. These new vectors all have five new unique 6-base restriction sites flanked by two 8-base restriction sites on both ends. The matching shuttle vectors have identical cloning sites to the transgenesis vectors, making the transfer of inserts from the smaller shuttle vectors into the larger transformation vectors much easier. In a proof-of-utility transformation experiment, the new pCaSpeR5 and pUAS-C5 transgenesis vectors were as effective as traditional pCaSpeR4 and pUAST in generating transgenic flies following standard embryo injection. The authors have provided Drosophila biologists with a cloning toolkit to facilitate simpler, easier cloning into the most commonly used fly transformation vectors. -Page 164
New Reporter for Mammalian Tissues
A wide selection of reporter systems employing a variety of detection schemes is currently available to analyze promoter function in prokaryotic and eukaryotic cells. Most reporters, however, are limited to use in cultured cells and in superficial tissues of large organisms due to the short path traveled by their optical signals. Ki et al. take an important first step toward developing a reporter for use in internal tissues of large living organisms with their demonstration that the product of Escherichia coli polyphosphate kinase (PPK) can be efficiently detected in cultured animal cells using 31P-magnetic resonance spectroscopy and 31P-magnetic resonance imaging. PPK catalyzes the synthesis—from ATP—of inorganic polyphosphate (polyP), which is not expressed at detectable levels in mammalian cells. The authors demonstrate that their PPK-based system performs well as a reporter when compared to luciferase, which is generally considered to be the most sensitive reporter. Further, this assay compares well with existing reporter systems in that it is nonin-vasive, quantitative, and appears to have no adverse effects on mammalian cells. Every indicator points toward the ultimate effective use of the PPK-based system as a reporter in internal animal tissues. -Page 209
Preimplantation embryos present unique challenges when analysis of gene expression by RT-PCR is the goal. Aberrant gene expression has been observed when comparing expression patterns of embryos produced by somatic cell nuclear transfer to those of embryos produced by in vitro fertilization, and there is controversy surrounding the use of at least one standard reference gene, histone H2A, as a control in gene expression studies in these embryos. In this issue, Bower et al. propose a universal method for normalizing gene expression data in preimplantation embryos. Plant-specific RNA and DNA spikes are added to the tissue prior to nucleic acid extraction. Following synthesis of cDNA, target gene transcripts and the exogenous RNA are assayed by quantitative RT-PCR and adjusted for cell number—made possible by the prior measurement of genomic copies of 18S-DNA by quantitative PCR and normalization of these values to the DNA spike. Use of an exogenous RNA as a pseudo-housekeeping gene in this manner permits quantitative gene expression analysis when only a small amount of tissue is available and when no appropriate endogenous reference gene is available. -Page 199
Promoter Analysis Is Flp-ing EasyAnalysis of promoter function traditionally relies on either transient transfection or stable integration of a promoter and reporter transgene in genomic DNA. However, both methods have drawbacks. Transient transfection introduces numerous copies of the reporter gene, but only a fraction of the constructs interact with the proteins needed for transcription, while stable integration relies on random integration of the promoter and reporter gene into the genome, making direct comparisons of mutant or polymorphic promoter regions difficult. Karimi et al. have devised a clever method to overcome these problems and examine promoter effects on gene transcription from a known chromosomal location. Using Flp recombinase-mediated integration, the authors were able to integrate a variety of promoters and a GFP reporter gene into a known genomic location and assay the efficiency of transcription using fluorescence-activated cell sorting. To further simplify the method, two vectors were constructed to make cloning promoter regions easier. As proof of the utility and potential of the method, the authors used the new method with the human complement receptor 2 (CR2) proximal promoter region and its known transcriptional silencer (CRS). Cells with only CR2 integrated had >90% fluorescence, while cells with both CR2 and CRS integrated into the Flp site were <10% GFP positive, showing strong transcriptional silencing of the CR2 transgene. Overall, this method will be extremely useful in the study of gene transcription. -Page 217
Show Me the Protein
The key to phage or bacteria protein display technologies is the ability to display as much protein as possible on the cell surface. Recently, several groups have utilized Bacillus subtilis spores for the purpose of displaying heterologous proteins. Isticato et al. demonstrate that the current Bacillus spore display method can be improved up to 5-fold with a simple modification. Fusion of a heterologous protein to a coat protein, CotC, directs the heterologous protein to the surface of the Bacillus spore for display. The authors found that fusion of the heterologous protein to the amino terminus of the CotC protein results in a dramatic increase in the amount of heterologous protein displayed on the spore surface. They validated this work by examining CotC fusions to both green fluorescent protein and the C fragment of the tetanus toxin and found that, in both cases, fusion to the amino terminus of CotC increases display efficiencies when directly compared to fusions to the carboxyl terminus. This simple modification will be useful for researchers using the Bacillus spore display system. -Page 151
Exon Arrays Arrive
Gene expression microarrays provide expression data at the level of the individual transcript and are now in wide use across life science research. The more recently introduced exon arrays have the capacity to provide expression information at the level of individual exons and will be particularly valuable in identifying expression patterns of alternatively splice variants of specific transcripts. In an early effort to assess the reliability of the new exon arrays, Okoniewski et al. compared the performance of the Affymetrix GeneChip® Human Exon 1.0 ST array to that of the HG-U133 Plus2 expression array using replicated RNA samples from two human cell lines. The authors describe the underlying design principles of the two types of arrays and explore three types of mapping strategy. They demonstrate a high degree of correspondence between results obtained with the exon and expression arrays, independent of the mapping strategy employed. Because the expression arrays have been validated comprehensively, these results strongly support the use of the exon array to obtain reliable gene expression data. -Page 181
