High-Resolution Imaging of Preimplantation Embryos

Fluorescent probes have become indispensable in developmental biology for the visualization of proteins and the study of cellular proliferation, differentiation, and morphogenesis. However, a drawback to this approach is that the high-intensity light required for laser-scanning confocal microscopy can damage the embryos. Two-photon fluorescence microscopy results in less phototoxicity, but is expensive and technically challenging. Ross et al. have described a method that uses spinning-disk confocal microscopy to image murine and bovine embryos at their most sensitive stage of development, the time of embryonic genome activation, which leaves the embryos viable. In fact, the imaged mouse embryos not only gave rise to healthy pups, these pups reached adulthood and generated their own healthy offspring (the researchers have to wait a little longer to determine if the same is true for the cows). The spinning-disk apparatus uses the same principle as laser-scanning confocal microscopy to obtain confocal images; in this case, however, the pinholes used to eliminate out-of-focus light are mounted on a spinning disk, which functions to merge the fluorescence into one uniform two-dimensional image. The authors hope to use their technique to correlate changes in fluorescently labeled markers over the course of development with later developmental competence of the embryo. This assessment of preimplantation embryos will be useful in experimental conditions that result in many nonviable offspring, such as somatic cell nuclear transfer. -Page 741

Cross-Linking Transcriptional Cofactors

The chromatin immunoprecipitation (ChIP) assay, essential for studying such processes as transcriptional regulation, DNA replication, and DNA repair, relies on the cross-linking of proteins to DNA. Formaldehyde has been the most commonly used cross-linking reagent for ChIP assays. Although it has been highly effective for identifying proteins that bind directly to DNA, such as histones and transcription factors, it has not been as effective for examining proteins that might be associated with the DNA but are not located in as close proximity to the DNA, such as transcriptional coactivators and corepressors. Therefore, Zeng et al. tested a number of other cross-linking reagents. Of the four they tested, EGS, the largest molecule with the longest spacer arm, was the most effective. Cross-linking with formaldehyde and EGS facilitated successful immunoprecipitation of LKB1, a p53 binding protein, from the p21/Waf1 promoter. This combination of EGS/formaldehyde was also capable of immunoprecipitating the GATA-1 cofactors FOG-1 and MTA-1 from the locus control region upstream of the murine -globin locus, while formaldehyde alone was ineffective. -Page 694

Monitoring miRNAs

MicroRNAs (miRNAs) are 20–25 nucleotide long stretches of noncoding RNAs that repress the translation of target mRNAs. They are usually tissue-specific and are often expressed in a developmentally regulated fashion. Thus, it would be very beneficial to be able to detect miRNA expression during cell fate changes in vivo. Unfortunately, the sensors used to detect miRNA expression often rely on negative data—the lack of translation of the sensor mRNA—so the presence of the miRNA must be confirmed. These sensors also require the generation of transgenic animals, which is laborious, expensive, and time-consuming. De Pietri Tonelli et al. have made a dual-fluorescence reporter/sensor plasmid that overcomes both of these limitations and allows the detection of miRNAs with cellular resolution in vivo. Their elegant system relies on a GFP reporter and an mRFP sensor, both under the control of identical constitutive promoters. Green fluorescence thus establishes the presence of the plasmid in a given cell, while red fluorescence indicates the lack of miRNA silencing. Using this technique, the group confirmed previous in situ data showing active miR-1 in zebrafish muscle fibers 33 h postfertilization. They also found that, contrary to currently held views, miR-124a is expressed not only in postmitotic neurons, but also in their progenitors, the neuroepithelial cells. -Page 727

Allele-Specific Methylation Analyses

Currently, there are two methodologies used to analyze DNA methylation. Bisulfite genomic sequencing of multiple alleles is labor-intensive, and bisulfite pyrosequencing does not assess allele-specific methylation levels. The latter is a severe limitation, as there are several instances when only one allele is methylated—for example, imprinting and X chromosome inactivation. Wong et al. have combined bisulfite PCR with allele-specific pyrosequencing to develop a rapid, easy, and quantitative method of allele-specific DNA methylation analysis. Like earlier methods, this approach is based on the principle that bisulfite deaminates cytosine, but not 5-methylcytosine, to generate uracil. The authors followed up bisulfite treatment with pyrosequencing, using primers directed to a heterozygous SNP, which were therefore unique to each allele. In a proof-of-principle experiment, they analyzed allele-specific methylation of the H19 gene, which is normally imprinted and methylated only on the paternal allele. Their new method correctly identified the percentage of allele-specific methylation within a 3% margin of error. This technique will be useful in epidemiological and tumor studies, for example, in identifying instances of loss of imprinting. -Page 734

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Introducing TEVcs

The tobacco etch virus (TEV) protease has been used as an in vivo tool to delineate the functional domains of proteins. It recognizes a rare seven amino acid sequence that can be inserted into a protein with few adverse effects because of its small size. Brown and Maloy have described a neat cloning trick that, for the first time, allows directed insertion of TEV cleavage sites (TEVcs) into defined sites; the approach can be used to test specific predictions about protein topology. They first used the λ Red recombination system to achieve the directed insertion of a selectable marker—in this case, chloramphenicol resistance. Next, they used the same system to replace this selectable marker with the TEVcs, which is nonselectable. The tricky part is that they also used an in-frame downstream reporter—the lac operon—to enrich for the desired replacement. This approach was effective because the Cam^R cassette included a strong transcriptional terminator to prevent the transcription of the downstream lac operon, which the TEVcs that replaced it lacked. They tested their method on the PutA protein from Salmonella enterica, an interesting choice because the crystal structure of the N terminus of PutA from Escherichia coli has just been solved. -Page 721

Extended BiFC

Bimolecular fluorescence complementation (BiFC) is a powerful tool for detecting protein-protein interactions in living cells. Proteins of interest are tagged at the N (YN) or C terminus (YC) with complementary fragments of yellow fluorescence protein (YFP); when an interaction occurs, the complementary fragments come together, producing a yellow fluorescent signal. However, this approach does not provide information on expression or localization of the individual protein partners, which would require additional analysis (e.g., FRET). Wolff et al. have modified the BiFC assay to provide expression and localization data in addition to interaction data. Proteins of interest, in this case the HIV Rev protein—a key regulator in HIV replication—and known Rev interaction partners were tagged with YN or YC, each fused to a second constitutively fluorescing domain—CFP (blue signal) or mRFP1 (red signal), respectively. Co-expression of pairs of interacting proteins in HeLa cells yielded blue, red, and yellow signals, identifying the free proteins and the interacting pairs; co-expression of non-interactors yielded only blue and red signals, despite colocalization of the proteins within the cell. This new approach, which the authors dub extended BiFC (exBiFC), adds an additional layer of information to BiFC analysis by normalizing the protein interaction to the expression levels of the individual interacting proteins and will surely extend the capabilities of BiFC. -Page 688