The Measure of a Man

If you want to get to know a person well, do not observe him in isolation, but watch him in the company of his family and friends. Likewise, if you desire to understand the significance of his genetic variability, do not simply type an individual SNP, but look to gain some context by determining a haplotype. This may be easier said than done: existing methods for molecular haplotyping are not well suited to the throughput requirements of today's genetic association studies. In a methods article appearing in a recent issue of Human Mutation, Xiao et al., describe a new measurement-based molecular haplotyping method in which multiple polymorphic sites on a single DNA molecule can be visualized simultaneously. First, long-range PCR (on the order of 10 kb) is performed. Then, padlock probes specific to each SNP to be interrogated are added to the PCR product. Single-base elongation with fluorescently labeled nucleotides is followed by ligation. The color of the fluorophore reveals the identity of the SNP, and the padlock arrangement of the oligonucleotide probe ensures that the fluorescent marker stays in place. Positional accuracy is vital because the DNA molecules are then stretched out on a glass coverslip and imaged by total internal reflection (TIRF) microscopy. TIRF enables extremely precise measurements, meaning that the nucleotide position of a fluorophore can be measured with sufficient accuracy to identify the corresponding SNP. By inspecting the color at each polymorphic position and performing data analysis of hundreds of molecules, the haplotypes of the sample can be inferred. The authors demonstrate the potential of their approach in a series of impressive proof-of-principle experiments. Though additional improvements in labeling efficiency and greater flexibility in fluorophore use are needed before this strategy is ready for routine use, the study implies that taking the measure of man in population genetic studies may simply entail a nanoscale measurement of labeled DNA.

- Xiao et al. 2007. Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding. Human Mutation 28:913-921.

Freedom of Assembly

In a developing organism, cells form intricate shapes corresponding to various tissues. It has long been recognized that standard cell culture conditions are a poor representation of the complex three-dimensional interactions that underlie these structures. More accurate mimicry of tissue architecture would improve in vitro modeling of normal development and disease states, most notably cancer. A new method for forcing cellular interactions, described by De Bank et al., uses the affinity between biotin and avidin for rapidly forming cell aggregates. The authors take advantage of the known ability of sodium periodate to generate aldehyde groups on cell surfaces, and then use these as covalent attachment sites for biotin. If avidin is added to a mixture of cells bearing these biotin molecules, the result is a network of biotin-avidin interactions that conspire to hold the cells in a multicellular aggregate. Not only do standard cultured cell lines such as NIH/3T3 put up with this kind of forced interaction, but the technique can also be used with embryonic stem cells. In the case of the latter, artificial embryoid bodies can be generated that replicate some of the differentiation pathways seen in traditional culturing methods, and may in fact offer an accelerated way of producing certain differentiated cell types. Another application of this surface engineering approach would be the formation of complex tissue-mimetic structures by organized aggregation of different cell types. As a first approximation of this, the authors show that a layer of fibroblasts can be arranged around a myoblast core. Though this is still some way from replicating the complex, multilayered structure of natural tissues, the ease with which the biotin-avidin interaction can be used to assemble cells will free bioengineers to experiment with new ways to imitate natural tissue formation.

- De Bank et al. 2007. Accelerated formation of multicellular 3-D structures by cell-to-cell cross-linking. Biotechnology and Bioengineering 97:1617-1625.

Going Green

Familiar to first-year biochemistry students the world over, RuBisCO may be the most well-known enzyme on the planet. As the catalyst behind the first step in photosynthetic carbon assimilation, RuBisCO is also notable as the most common protein in nature. Unfortunately, for all its impressive credentials, RuBisCO does not have particularly favorable kinetic properties. Tweaking its efficiency might facilitate the development of genetically engineered plants that are able to use water and nitrogen more effectively, and may ultimately improve crop yield. In order for enzyme engineering of RuBisCO to be practical, an easy, high-throughput assay of enzyme activity is vital. Such an assay would also benefit plant cell biology studies, for some important aspects of RuBisCO's regulation are incompletely understood. The most commonly used assay for RuBisCO activity is a time-tested method that uses ¹⁴C-labeled carbon dioxide; however, the use of radioactivity and the rigorous requirements for buffer preparation make it relatively inconvenient. Hence, Sulpice et al., were motivated to develop a nonradioactive method that can be performed in a standard 96-well format. The technique is based on absorbance measurements and thus requires no equipment beyond a microplate reader (and pipeting robot, if desired). Only small amounts of extract are needed; they can be stored in a freezer prior to assay with no loss in measured activity. As a taste of the potential of this assay, the authors describe preliminary experiments to identify genes that may be involved in the regulation of RuBisCO. To do this, they characterized the enzymatic activity of a number of Arabidopsis variants and looked for associations with particular genetic markers. Although no associations were confirmed, the throughput of the assay (on the order of several hundred assays per day) is clearly better suited to large-scale genetic studies than the previous method. With its ease of use and many potential applications, this new RuBisCO assay may ultimately become nearly as ubiquitous as the famous enzyme itself.

- Sulpice et al. 2007. Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant, Cell and Environment 30:1163-1175.

Special Delivery

Polarized epithelial cells can have impressive secretory abilities, but that doesn't mean that on occasion it isn't important to introduce molecules into them instead. Just as with other cell types, the ability to transfect these cells with DNA or proteins can reveal critical information about cellular regulation and behavior. Unfortunately, it is an inconvenient fact that transfection of polarized epithelial cell lines such as MDCK or Caco-2 is not particularly easy. Existing lipofection and electroporation techniques are not designed for efficient delivery into confluent epithelial monolayers. Although these cells make good targets for microinjection, such a laborious and time-consuming method is undesirable for routine transfection. Similarly, adenoviral vectors, while efficient, may be tedious to prepare by cloning, and suitable receptors may not be available on all cell types. Faced with these unsatisfactory alternatives, Deora et al., went back to the drawing board to develop an optimized electroporation procedure. The resulting protocol is designed for polarized epithelial cell lines or primary cultures that are grown on transwell filters and uses a commercially available electroporation device. Transepithelial resistance measurements showed that tight junctions recovered as quickly as 1 h post transfection. Importantly, faithful targeting of proteins to the cell surface appeared to be preserved, as plasmids encoding tagged proteins destined for apical or basolateral membranes or the trans-Golgi network were all appropriately trafficked. The technique showed a 20-fold improvement over transfection attempted with popular lipofection reagents, and also allowed transfection of fluorescently labeled antibodies. Although intended to facilitate study of polarized epithelium, these conditions may prove useful for other systems in which comparatively gentle electroporation of confluent, differentiated cultures is necessary.

- Deora et al. 2007. Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture. Traffic [Epub ahead of print, July 27, 2007].