Some Like It Hot: Improved 454 Library Recovery

Next-generation sequencing technologies such as the 454 platform have significantly increased the number of basepairs that can be sequenced per run. However, when the amount of template DNA is very small (for example, that from paleontological bone samples), the number of DNA sequences that can be determined can be limited. In order to maximize DNA sequence determination from such samples, T. Maricic and S. Pääbo at the Max Planck Institute for Evolutionary Anthropology (Leipzig, Germany) analyzed the recovery of template DNA from each of the five major steps of 454 library preparation in order to determine which ones required optimization. While the first four steps had reasonable recoveries of 40–96%, the last step, involving NaOH denaturation to obtain the single-stranded library from the biotinylated-adapter-ligated double-stranded DNA bound to streptavidin beads, resulted in losses of over 99%. When they replaced the NaOH denaturation with a heat denaturation by incubation at 90°C, recovery increased to 98%. This also has the advantage of recovering both strands of template DNA bound to the streptavidin beads, allowing the determination of base changes that occur on only one strand—such as miscoding lesions in ancient DNA—that may be missed by using NaOH denaturation, which recovers only one template strand.

(See “Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands” on page 51.)

Ubi or Not Ubi, That is the Fusion

The generation of stably transfected mammalian cell lines expressing an ectopic protein typically requires selection for an antibiotic resistance gene expressed from the same construct as the protein gene. While the preferred vectors for this are currently those with an internal ribosome binding site (IRES), which allow translation of both genes from the same transcript, the translation rates of the two proteins may differ significantly. K. Matentzoglu and M. Scheffner at the University of Konstanz (Konstanz, Germany) have developed a vector that ensures that the antibiotic resistance and transgenic proteins are generated in equimolar amounts, since they are derived from the same polyprotein. They fused the puromycin resistance gene (puro^r) to the N-terminus of ubiquitin (ubi), which can then be fused to the N-terminus of the protein of interest in their vector. The resulting polyprotein is cleaved by ubiquitin-specific proteases (USP) to release the puro^r-ubi fusion protein and the protein of interest, and they confirmed that the puro^r-ubi fusion protein conferred puromycin resistance to transfected cells. Additionally, they observed that the level of expression of the protein of interest directly correlated with puromycin selection pressure. This ubiquitin fusion protein expression system expressed five to ten times the amount of protein from IRES-based constructs.

(See “The ubiquitin-fusion protein system: a powerful tool for ectopic protein expression in mammalian cells” on page 21.)

Cells in Gels Go Undercover

Cells encapsulated in hydrogel spheres are used for various applications. For example, encapsulation of the insulin-producing islets of Langerhans isolates them from the immune system and cellular environment while allowing trafficking of insulin, nutrients, and byproducts. This method is limited, however, since cells completely encapsulated in hydrogel are not adherent and cannot interact with signaling molecules in the extracellular matrix that are crucial for cell survival, proliferation, migration, differentiation, and apoptosis. M. Gepp and colleagues from the Fraunhofer Institute for Biomedical Engineering (St. Ingbert, Germany) have developed a new device and method for dispensing high viscosity alginate in low volumes to encapsulate adherent cells of interest. Cells encapsulated by this technique showed up to 90% viability, successfully completed mitosis at a rate similar to that of non-encapsulated cells, and displayed healthy physiological functioning as evinced by motility, metabolic activity, and gene expression. Cells within the alginate contacted their substrate and exchanged chemical signals, but were still isolated from potentially damaging macromolecules in their environment. This system provides a ready means for generating encapsulated adherent cells and structuring substrates for biotechnology and regenerative medicine applications.

(See “Dispensing of very low volumes of ultra high viscosity alginate gels: a new tool for encapsulation of adherent cells and rapid prototyping of scaffolds and implants” on page 31.)