Co-detection of Dystrophin and GFP in Sections

Absence of dystrophin, a cytoskeletal protein, produces the progressive muscle degeneration of Duchenne muscular dystrophy (DMD). Introducing dystrophin-producing cells (derived from muscle or other tissues) into the diseased muscle may help alleviate the condition. Labeling and tracking dystrophin-positive cells is important to evaluating the potential performance of these engraftments. Researchers commonly label donor cells with GFP to facilitate tracking and characterizing them and their offspring in tissue sections. GFP expression is suggestive but not sufficient to evaluate the therapeutic potential of donor cells, unless it is demonstrated that donor cells following fusion with pre-existing myofibers also express dystrophin. Still, most studies detect either GFP alone or assay for GFP and dystrophin separately, in different tissue sections. Liadaki et al. report in this issue on a method for simultaneously tracking both GFP fluorescence and dystrophin expression in the same muscle section. They isolated tissue samples showing GFP activity in the myofibers and treated them for subsequent labeling with rabbit polyclonal anti-dystrophin antibodies, followed by staining with an anti-rabbit stain. The method, say the authors, allows efficient use of rare materials and adds precision to the evaluation of different cell populations’ therapeutic potentials. -Page 699

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New Looks for Familiar Stains

The Dark Side

In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. Trinh et al. demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence (excitation 633-nm laser stimulates emissions collected with a 650-nm long pass filter) that allows confocal microscopy to generate high-resolution three-dimensional imaging of gene expression. The method permits cellular resolution in whole-mount in situ hybridization studies. The red excitation and NIR emissions are ideal for use in whole-mounted specimens as longer wavelengths are less scattered by tissue. Autofluorescence is much lower in this wavelength range in most specimens, according to the authors, who add that NIR emission should facilitate multilabel NBT/BCIP staining protocols in combination with visible fluorophores—without development of new protocols for fluorescent in situ hybridization methods. -Page 756

The Light Side

In another paper in this issue, Jékely and Arendt examine performance of the same NBT/BCIP precipitate to profile cellular expression in three dimensions using reflection confocal microscopy. Until now, the most sensitive and widely used staining technique for whole-mount in situ hybridization (WMISH), NBT/BCIP precipitation by alkaline phosphatase, could not be combined with confocal laser-scanning microscopy (CLSM). The authors now report efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. Whole-mount reflection CLSM should, the authors say, facilitate large-scale, cellular resolution expression profiling in vertebrate and invertebrate model organisms. -Page 751

Inflammatory Supports

Some common cell culture support materials can trigger inflammatory responses in macrophage-like U937 cells. Matheson et al. studied the functional parameters of differentiated U937 cells cultured on tissue culture-grade polystyrene (TCPS) and polydimethylsiloxane (PDMS). Both surfaces elicited enzyme markers of inflammation. Cells cultured on TCPS showed higher levels of intracellular and released esterase, whereas cells raised on PDMS produced more phosphatase and released protein markers. Researchers evaluating inflammatory responses do not sufficiently appreciate the impact of support materials on cell function, the authors warn, and should take care to evaluate these background effects when screening drug candidates or undertaking studies to elucidate metabolic pathways. -Page 744

Antibody Phage Display Using Bacterial Cells Bearing Helper Phage Plasmid

Phagemid particles for phage display are conventionally made by superinfecting phagemid-containing cells with helper phages. Shi et al. reasoned that they might produce phagemid particles more efficiently by constructing a host bacterial strain containing a helper phage plasmid into which phagemids could be transformed. Since production of protein III (pIII) by host cells can mediate resistance to filamentous phage infection, the researchers utilized a helper phage plasmid with a pIII deletion mutation. In this month's Research Report, they describe their novel method of pIII-based antibody phage display using their host bacterial strain Hpd3cells (E. coli TG1 cells transformed with the pIII-deleted VCSM13d3 helper phage plasmid). This method improves on previous hyperphage display methods in ease of use, and yielded phagemid particle titers of 1.2 × 10¹¹ cfu/ml, comparable to previously reported yields from conventional methods. The authors suggest that Hpd3cells should allow efficient enrichment of specific binding antibodies from a phage display library, and can be used for the single-chain antibody fragments (scFv) reported here, as well as for high-level ligand or antigen display. They also suggest that the approach might be extended to modify other capsid genes. -Page 760

More ShRNAs for Embryonic Stem Cells

An improved system for generating short hairpin (sh)RNAs for loss-of-function studies, pHYPER, is four times as active as its predecessor system, the pSUPER vector (based on a 0.2-kb H1 promoter). The new system combines a 2.5-kb mouse genomic fragment (incorporating the H1 promoter), which provides consistent results in embryonic stem cells. The system is designed to support both transient transfection studies and long-term stable stem cell lines. Berlivet et al. report on a continuing effort to optimize shRNA technology in embryonic stem cells by testing various constructs derived from the H1 promoter. -Page 738

Yeast Two-Hybrid Screen for Transcriptionally Active Proteins

Understanding all possible protein-protein interactions in a living cell is essential to understanding biological processes. Möckli et al. extend the range of available tools for examining these interactions with a novel genetic screening assay, the cytosolic yeast two-hybrid system (cytoY2H), based on the split-ubiquitin technique, one of the most widely used protein complementation approaches. The new cytoY2H assay combines the split-ubiquitin assay with a stringent transcriptional output using multiple independent reporter genes to detect protein-protein interactions in the cytoplasm. It can be applied to study a wide range of proteins that are difficult to detect in the classical yeast two-hybrid system, including transcription factors like p53 (which autonomously activate transcription and so defeat the assay) and members of the NF-κB complex. In this work, the authors applied the cytoY2H system to cDNA library screening and identified several new interaction partners of Uri1p, an uncharacterized yeast protein. -Page 725