In this Issue

Delivering siRNAs

Implementation of the phenomenon of RNA interference as an experimental tool is fraught with technical challenges, including transfection of cells with biologically active RNA. Gruber et al. (p. 96) have adapted the process of osmotic lysis of pinosomes, traditionally used primarily for introduction of proteins and dyes, to deliver siRNA (short interfering RNA) into mammalian cells. Using fluorescently labeled siRNA and confocal microscopy, the authors show that siRNA in hypertonic medium is taken up by a variety of cell types by the process of pinocytosis and that the siRNAs are released from pinosomes into the cytoplasm when cells are subsequently “shocked” by treatment with hypotonic medium. They obtain silencing efficiencies of 60%–90% using the process that is dependent on uptake time and siRNA concentration. This novel approach provides a valuable alternative to existing techniques for transfecting cells with siRNA.

Timing is Everything

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a robust platform for protein profiling and differential gene expression analysis. The approach relies on the capture of analyte—proteins and peptides—by chemically defined arrays following deposition of crude sample and matrix—small UV-absorbing molecules that transfer energy from a laser source to bound analyte for desorption and ionization. In an investigation of parameters that influence spectral quality and reproducibility, Jock et al. (p.30) investigated the effect of the length of the time interval between sample application and matrix deposition on the array. They identified significant differences in spectral properties related to timing that were specific to conditions of array type, buffer, and matrix composition. This study is a useful examination of quality control parameters and will contribute to the continuing refinement of SELDI-TOF MS as a tool for proteomics analysis.

Lockdown

Graduate student grumbles aside, most investigators have little in common with prison guards. Researchers who genotype using Pyrosequencing, however, may soon discover the benefits of lockdown—of errant priming, that is. The trouble derives from the Pyrosequencing reaction conditions, which can permit the template to fold back on itself and inappropriately self-prime. Although there are some strategies to try and prevent or lessen the problem, none has been wholly satisfactory. Stepping up to the challenge, Utting et al. (p. 66) investigate two methods to prevent self-priming. In the first approach, a blocking oligo, complementary to the 3′ end of the template and ending in a dideoxynucleotide, is used to compete with the self-priming reaction. The second strategy uses terminal deoxynucleotidyl transferase to treat the 3′ end of the template so that it cannot be elongated. The authors show that both strategies prevent ghost peaks and reduce background, allowing more confident sequence interpretation and more accurate quantification of allele frequencies in genotyping.

A Pox on HIV

Vaccination made its debut with a pox virus, so it's not surprising that these DNA viruses are still being evaluated for immunostimulatory potential. Recombinant pox viruses are appealing for expression of antigens and/or immunomodulatory proteins because they can accommodate longer coding sequences than trimmer retroviral vectors. Avipox viruses, like fowlpox virus, would be of particular interest as vaccine candidates, as their inability to replicate in nonavian cells provides added safety. Unfortunately, for all of their advantages, fowlpox viruses are not easy to engineer. Boyle et al. (p. 104) describe plasmid transfer vectors for production of recombinant fowlpox virus (rFPV). These new tools ease the construction of rFPV, making the process comparable to working with vaccinia or adenovirus recombinants. Significantly, the new transfer vectors permit transient dominant selection, enabling construction of rFPV that are free of extraneous foreign DNA sequences. To show the value of their tools, the authors generate an rFPV containing three human immunodeficiency virus (HIV) antigens, now a vaccine candidate destined for a phase I clinical trial.

Tissue-Targeted Therapy

Phage display has become an important tool for obtaining peptides with defined binding specificities and has been used in vivo to identify peptides that bind to tissue-specific ligands. While many reports exist on the targeting of ligands in adult animal tissues, there have been no published studies of the targeting of fetal tissues by phage display using the maternal systemic circulation. Srivastava et al. (p. 81) inferred that such an approach could help correct many genetic and metabolic diseases if applied in utero. They demonstrated the distribution and detection of a T7 phage peptide library in mouse fetal tissues after systemic injection into tail veins of pregnant mice. Such an approach would be sure to find application for the design of in utero tissue-targeted therapies.

Normalizing RNA Expression

Normalizing RNA levels to internal or housekeeping reference standards is widely used to control for sample error when measuring RNA expression levels. Choice of an appropriate reference standard is critical to avoid misinterpretations of results, and reference validation is crucial in assessing the utility of a particular experimental model. Dheda et al. (p. 112) report a validation protocol to identify the most suitable housekeeping standards in studies of pulmonary tuberculosis. They used real-time reverse transcription PCR (RT-PCR) to study the levels of 13 housekeeping genes expressed in blood cell cultures of groups of both healthy and tuberculosis patients. Only one gene, HuPO, was found to be suitable for normalization of RNA levels for these studies, unlike GADPH and β-actin that were each found to be highly variable. Their results show that housekeeping genes can be variable and prone to shifts in expression induced by experimental conditions, thereby causing problems for reliable normalization. In order to avoid significant inaccuracies when interpreting RNA expression data, this study emphasizes the importance of validating controls for individual experimental systems.

Aptly Used Aptamers

The majority of annotated genomic sequences have no available corresponding cDNAs. Those that exist do not often include all possible splice variants. For direct synthesis of cDNA clones that correspond to predicted genes, new splice variants, or other genes of interest, Mitani et al. (p. 124) developed a method to effectively amplify exons from genomic DNA. The novelty of their approach is the use of a Taq aptamer that allows specific amplification of the exons, necessary for the success of the second PCR, in which exon assembly takes place by overlap extension. The authors validated their technique by synthesizing the 1.9-kb full-length open reading frame (ORF) of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene in a two-step reaction involving all 13 exons. This method will allow a more rapid completion of in vitro synthesis of cDNAs and alternative splice variants from genomic DNA and can be applied as well to targeted gene manipulation.

Negating False Positves

High-throughput screening can slow down very quickly if artifacts begin obscuring the legitimate hits. Mindful of this concern, Cunningham et al. (p. 120) set about to identify agents that nonspecifically up- or down-regulate reporter plasmids in cell-based screening assays. The analysis stemmed from the authors’ previous screening work, which had pulled out a histone deacetylase inhibitor when, in reality, they were looking for compounds with specific effects on Smad4 function. To find other false positives, the team screened over 17,000 agents from compound and drug libraries and identified 14 candidate nonspecific enhancers. These agents were then tested with other reporter plasmid systems to study the extent of their activity, and initial studies were performed to get hints about possible mechanisms of action. All told, this information will be of interest to investigators assessing results from screens using cell-based reporter assays as they work to separate the wheat from the chaff.