Currently, RNA interference (RNAi) seems to be the technique of choice for knocking down gene function in vivo. Although appearing simple at first glance, effective gene knock-down by RNAi can be complicated by numerous factors, from the design of a good small interfering RNA (siRNA) to its delivery in the cell. In the current issue, Morlighem et al. describe a new assay to rapidly examine efficacy of siRNAs, providing a method to determine whether or not a particular siRNA will effectively silence the target gene. The new assay is based on fusing a reporter gene to the target gene and assaying for decreases in the activity of the reporter gene. The authors used human p53 as the target gene fused to the GLuc reporter, a secreted Gaussia luciferase gene. It turns out GLuc is a very useful reporter for this assay since it is secreted, and therefore the authors were able to assay reporter gene activity using fluorescence measurements in a microtiter plate, allowing numerous siRNAs to be tested together. In a proof of utility experiment, five synthetic siRNAs for human p53 were compared to one RNAse III-generated siRNA mixture. It was shown that the siRNA mixtures tested resulted in effective knock-down (up to 85%), whereas only a single synthetic siRNA demonstrated comparable results. The authors validated the findings from this new assay with comparisons to quantitative real-time PCR and Western blot data, confirming reductions in the levels of both messenger RNA (mRNA) and protein. This new assay should prove extremely helpful for the rapid selection of potent siRNAs. -Page 599
Tissue Pre-EmbeddingHistologists often arrange tissue samples in a very specific orientation prior to paraffin embedding using a process known as pre-embedding. Pre-embedding has traditionally utilized molten agar that is poured over the tissue and which hardens to maintain the proper orientation of the tissue during subsequent embedding procedures. A shortcoming of using agar as the pre-embedding media is that certain tissues shrink during the embedding process, and the agar-based pre-embedding media limits tissue expansion during slide mounting, resulting in difficulties with the tissue sample adhering to the microscope slide. Jones and Calabresi have provided a simple solution to this challenge by using agar mixed with gelatin as a new pre-embedding media. The authors demonstrate that for central nervous system tissues, which tend to shrink during embedding, using agar-gelatin pre-embedding allows these tissues to completely expand when placed on a 42°C water bath prior to slide mounting. The gelatin is thought to melt away at 42°C, providing space for the shrunken tissue to rehydrate and expand. This simple modification to the pre-embedding process will help histologists pre-embed all types of tissues, preserving the needed orientation prior to paraffin embedding without sacrificing proper tissue adherence to a microscope slide. -Page 569
The use of chemical genetics for drug discovery and elucidation of protein networks in cells has exploded in the past few years. The ability to identify compounds that block protein interactions in the cell has the potential to not only identify novel drugs, but also further our basic knowledge of the biology of the cell. In this issue, Joshi et al. describe a new modification to the repressed transactivator (RTA) yeast two-hybrid screen that permits small molecule screening to identify inhibitors of protein binding. RTA relies on a transcription activator as the bait that, in the absence of binding to the prey that fuses to the Tup1RD repressor, results in expression of a URA3 reporter gene and sensitivity to 5-fluoroorotic acid (5-FOA). To permit forward selection, a HIS3 reporter gene was incorporated allowing selection on 3-aminotriazole (3-AT). Resistance to 3-AT therefore occurs when bait and prey cannot bind due to inhibition. To test the new system, the authors initially examined interactions between FKBP12, a protein that regulates intracellular calcium mobilization, and its binding partner, the TGFβ receptor. FKBP12 binding to the TGFβ receptor is disrupted by several chemicals, including FK506, ascomycin, and cyclohexamide. Using the modified RTA system, the authors found these drugs resulted in resistance to 3-AT, indicating disruption of binding, but to varying degrees. This showed that not only does the RTA system have the potential to identify drugs that block protein interactions, but the degree of resistance to 3-AT also is indicative of the affinity of the proteins for the molecule. The authors then performed a chemical screen using a 23,000-compound small molecule library to identify novel compounds able to disrupt FKBP12 and TGFβ interactions. Four novel molecules were identified from the screen, which were validated in vivo. This modification to the RTA yeast two-hybrid system will allow high-throughput screening for inhibitors of protein interactions and at the same time provide novel insights into basic cell biology. -Page 635
Bacteria can aggregate on surfaces forming microbial communities known as biofilms. The study of biofilms presents a great technical challenge for microbiologists since visualizing a three-dimensional microscopic world can be difficult. Expensive microscopy methods, such as scanning electron microscopy and confocal microscopy, have been used to meet the challenge of looking at these three-dimensional assemblages of bacteria, but less expensive methods would be a welcome addition to this growing area of microbiology. de Carvalho et al. present a new method to study the size and composition of microbial biofilms using only bright-field microscopy. The idea presented by the authors is that the intensity of each pixel in the x-y plane, obtained using a bright-field microscope, is an indication of the number of cells in the z plane. To determine this relationship experimentally, the authors studied frozen biofilms where the cells had been stained. These stained cells were then observed under fluorescent light to determine the exact number of cells for a given intensity of light. This relationship was then applied to the data generated from the bright-field microscopic observations. In a proof-of-utility experiment, the effect of solvents on biofilm formation was assessed, demonstrating that the new method using bright-field microscopy is effective at assessing overall biofilm size. This new method should prove useful for scientists wanting to study biofilm formation without the need for expensive microscopy equipment and analysis. -Page 616
Chromosome Deletions: Where Less Says More
The ability to generate large chromosomal deletions and assay the phenotype in cells can provide valuable information regarding genes located within the deleted region. Generating these large chromosomal deletions can be both a difficult and time-consuming process. Zhu et al. describe a new system for the efficient generation of random chromosomal deletions and translocations. The new method relies on the use of the cre/LoxP system to cause the recombination and antibiotic resistance markers to select for cells where either deletions or translocations have occurred. To accomplish this the authors first divided the puromycin resistance gene into two parts, fusing a single LoxP site to each, and subsequently cloned the two parts into separate retroviral vectors that could be transfected into cells. Following transfection of both parts of the divided puromyocin resistance gene, cells could be selected on the basis of puromycin resistance, an indication that recombination occurred between the two LoxP sites to generate an intact puromycin resistance gene. Using the new system, the authors describe the generation of a wide range of chromosomal deletions, from approximately 4 million base pairs to almost 20 million base pairs in size. This new method provides researchers a simple set of tools to generate large chromosomal deletions rapidly and effectively. -Page 572
