Obtaining pure organ preparation by microdissection from small organisms, such as the zebrafish, Dario rerio, is itself a sufficiently arduous task. Isolating such organs in the same manner from the fish in its embryonic stage is near impossible. With this technical hurdle to mount, Burns et al. developed a simple and straightforward isolation technique, described on p. 274 of this issue, which yielded good quantities of almost homogeneous zebrafish heart. The basic setup included a fine, large-gauge needle and syringe, clamped securely over a microfuge tube containing the zebrafish embryos (those between and 2 and 4 days postfertilization were used). Making use of the shear forces created by repeatedly drawing up and expelling the solution containing suspended embryos, the yolk sac could be broken, and the embryos fragmented. Filtering through two grades of nylon mesh generated a highly pure suspension of zebrafish hearts. The procedure was sufficiently vigorous to produce a good yield, but also gentle enough that intact organs were obtained that, when warmed to room temperature in media, demonstrated spontaneous rhythmic contractions. Further proof of the purity of the extract and success of the technique was provided through tissue-specific quantitative RT-PCR analysis.
Thinking Outside the BOXTO
The development of new, brighter, and more stable fluorescent molecules and DNA dyes covering a broader emission range has dramatically increased the flexibility for experimental design available to scientists. Multicolor fluorescent in situ hybridization, more complex fluorescence resonance energy transfer (FRET) setups, and enhanced multiplex real-time PCR are just some examples. Lind et al., on p. 315 of this issue, describe one such case of increased flexibility, in which they make use of a recently developed dsDNA binding dye for melting curve analysis in conjunction with established real-time PCR methodologies with the commonly used FAM fluor. The dye, BOXTO, a member of the BEBO family of groove-binding cyanine dyes, was developed for use in real-time PCR applications as a potential alternative to SYBR® Green. Now, the authors further extend the application of BOXTO by showing that it can be used in combination with sequence-specific FAM-labeled probes. While the latter are adept at detecting only targeted PCR product, post-PCR melting curve analysis using BOXTO can identify the presence of aberrant products such as nonspecific products and primer-dimers. Since the emission wavelength of BOXTO is sufficiently different from that of FAM (552 and 521 nm, respectively), their signals will not overlap, enabling facile detection of both in the same reaction tube. BOXTO did not significantly alter the efficiency of the PCR, although a small affect on the CT value was seen.
Totally TubularSingle-walled carbon nanotubes are increasingly employed in biomedical research; these applications frequently require f unctionalization of the nanostructures. In a report on p. 295, Didenko and Baskin describe an enzymatic approach for labeling nanotubes with quantum dots. The strategy is based upon the tyramide-horseradish peroxidase reaction, in which the enzyme reacts with tyramide conjugates to generate radicals that can mediate attachment to a substrate of choice, in this case the nanotube. Unfortunately, using a tyramide-fluorophore conjugate does not result in labeling, as the nanotube quenches the proximate fluorophore. Instead, the authors performed the reaction with a tyramide-biotin conjugate and mixed the resulting biotinylated nanotubes with streptavidin-linked quantum dots. They found that the extent of labeling could be determined by agarose electrophoresis, as nanotubes, unlike free quantum dots, cannot enter the gel. Electron microscopy revealed quantum dots decorating the nanotubes in a “beads-on-a-string” configuration. In fluorescence microscopy, ropes and individual nanotubes could be observed. This functionalization method should allow straightforward production of labeled nanotubes that can be applied to the study of single molecules; in addition, the technique provides a general strategy for bioconjugation of single-walled carbon nanotubes.
The Other Terminator
As those of us who have in the past had one of those forehead-slapping moments will attest, sometimes the answers (or car keys) are right in front of our faces. The paper from Tebbutt et al. on p. 331 appears to be a case in point. Coaxed on by the extraordinarily high cost of dideoxynuclotides (ddNTPs), potential licensing complications involved in their use, and a paucity of suitable fluorescent labels, the authors investigated their substitution with regular deoxynucleotides (dNTPs) in microarray minisequencing reactions. Using arrayed primer extension, or APEX, to genotype two sets of human DNA samples, Tebbutt and his colleagues determined that fluorescently labeled deoxynucleotides worked almost as well as equivalent dideoxynucleotides, with only a small loss in accuracy and sensitivity. This allows for potentially significant savings in consumables cost for any laboratory routinely performing SNP genotyping with APEX or similar dideoxy terminator reactions. Further, the use of dNTPs provides a wider range of compatible fluorescent labels, increasing the flexibility of experimental conditions for multiplexing reactions. Exactly why reactions terminate using dNTPs when there is still a reactive hydroxyl group available to the polymerase is still undetermined; the authors hypothesize that stearic hindrance might play a role, but additional experiments are clearly necessary to fully explain this usual, but plainly useful, phenomenon.
Control Freak
In order to produce host-toxic recombinant proteins in E. coli, tight regulation of expression is required. One such tightly regulatable vector is pBAD, which relies upon catabolite repression and positive induction; however, this control comes at the cost of production of only a relatively small amount of recombinant protein. Another alternative is a vector system in which the recombinant protein is under the control of T7 polymerase, which is itself expressed from a lactose-inducible promoter located on the bacterial chromosome. In this case, higher-level expression is achieved, but with a disadvantage that the system is prone to leaky expression. A system that combines tight regulation with high-level induction would be enormously useful; on p. 355, that is just what Giacalone et al. describe. The new vectors are induced by L-rhamnose and repressed by D-glucose. The authors show that their pRHA vectors are capable of efficient protein production upon induction and that the level of recombinant protein produced is proportional to the amount of inducer added. Importantly, the pRHA construct permits production of a toxic GPCR chimera that induces growth arrest in pBAD- and pET-based expression systems. This new rhamnose-inducible protein expression system should be of broad applicability in high-level expression of proteins for functional assays or structural studies.
Brain Gains
Many insights about neurotransmitter release have resulted from the direct visualization of vesicle release and uptake in presynaptic terminals. Historically, such analyses have been performed in primary neuronal cultures. Brain slices are believed to offer a more physiologically relevant system; however, these preparations tend to attract nonspecific binding of the FM® styryl dyes that are typically used in vesicle tracking. This frustrating problem can be partially addressed by the use of quenching compounds, but these chemicals can interfere with measurements of dye uptake into nerve terminals. In a compelling study, Winterer et al. (p. 343) show that two-photon laser-scanning microscopy (TPLSM) permits ready discrimination of specific vesicle-associated signal from background fluorescence. In particular, this method results in improved lateral resolution of fluorescent puncta by TPLSM as compared to confocal laser-scanning microscopy. Thus, TPLSM represents a robust method for monitoring ongoing changes in the total and readily releasable vesicle pools.
