Cells in the Real World

PloS One represents a novel concept in journals, offering rapid peer-review, online publication, but most intriguing is the ability for everyone to comment upon and annotate papers, making the publication process more dynamic and interactive. In the inaugural issue of PloS One, (Gelain et al). describe a cell culture system that allows neural stem cell differentiation using self-assembling peptide nanofibers as scaffolds. A common problem with tissue culture systems is the inability to recapture the in vivo environment. Reconstruction of a 3-D environment on a standard Petri dish or a microwell plate has been difficult to achieve. The authors build upon earlier work using the self-assembling peptide RADA16, which forms nanofiber scaffolds capable of supporting cells in a 3-D environment. Here the authors demonstrate that longer RADA16 peptides containing additional functional motifs for cell adhesion, differentiation, and bone marrow homing still form functional nanofiber scaffolds. In addition, using neural stem cells, two modified scaffolds containing bone marrow homing motifs could direct growth and differentiation toward cells expressing neural and glial markers without the need for externally provided growth or neurotrophic factors. Interestingly, cells in this environment have different transcriptional profiles of extracellular matrix and adhesion genes when compared to cells grown in standard culture dishes on an artificial matrix. These differences could reflect the interactions necessary between cells and the synthetic scaffolds of RADA16 nanofibers. In comparison to standard methods, the use of these “designer peptide nanofiber scaffolds” provide investigators with a more defined experimental environment and the ability to make single amino acid changes in introduced motifs—a level of modification impossible using current growth media. This new method appears very promising for creating cell culture systems that can better mimic the in vivo world cells inhabit. Over time it will be fascinating to see what changes, additions, and modifications to this new system take place; eventually these might be found annotated and attached to this paper at PloS One. –NB

-Gelain et al. 2006. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PloS One 1:e119.

Metagenomics Tackles the Termite

Attempts to identify individual microbial species from complex environmental samples have become increasingly commonplace in biological research. However, most methods, including PCR-based and computational whole-genome sequence assembly, have resulted in limited, at best, classification of specific microorganisms from such samples. Now (Ottesen et al). introduce a new method to obtain the identities of microbial species in these community structures using the technology of microfluidic digital PCR. The complex environment the authors explore is the hindgut of the wood-feeding termite wherein symbiotic microorganisms produce acetate, a major energy source for the termite. Termite hindgut luminal contents were placed into buffer and loaded onto the microfluidic device, which partitioned single bacterial cells from the sample into 1176 independent 6.25-nl PCR reaction vessels. Multiplex PCR was performed using probes to the formyl-tetrahydrofolate synthetase (FTHFS) gene, a key gene in the acetate synthesis pathway, and 16s rRNA, which is used for phylogenetic classification of microorganisms. Using this system, the authors show that only a small subset, approximately 1% of the hindgut symbionts, possess the FTHFS gene and therefore carry out acetate synthesis. Further analysis of 16s rRNA sequences revealed that these bacteria were all affiliated with the Treponeme cluster of Spirochaetes, demonstrating that several closely related Treponema species are responsible for acetate production in the termite hindgut. This work demonstrates the potential of microfluidic digital PCR to identify specific microorganisms from more complex parental samples. Although preliminary, this is an elegant step forward in defining the functions of different microorganisms within complex communities. –NB

-Ottesen et al. 2006. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314:1464-1466.

Elegant Engineering in elegans

Although C. elegans has been an extremely popular model organism to study everything from development to aging, tools for genome engineering in worms remain limited. Homologous recombination-based approaches have been stymied by the inefficiency of recombination between exogenously introduced DNA fragments and the C. elegans genome. To coax homologous recombination to occur at a particular locus, researchers working in other systems have employed a transposon-induced double-strand break (DSB) strategy. Unfortunately, endogenous transposons in the worm are repressed in the germline except in “mutator” strains, where there is a high risk of ending up with additional, undesired mutations. Clearly, an inducible, heterologous transposon would be a useful tool for introducing DSBs in a controlled manner. The Drosophila transposon Mos1, which can be controlled with a heat-shock inducible Mos transposase cassette, has been previously used for insertional mutagenesis and mutation mapping in worms. Here, (Robert and Bessereau) describe how Mos1 can be used to trigger DSB repair of targeted chromosomal breaks. Repair can involve end-joining (which may or may not rescue the Mos1-interrupted gene) or, as desired, can cause gene conversion when a transgene is used as the template for homologous recombination. In a series of tests, Robert and Bessereau demonstrate that gene conversion occurs at a frequency of 10⁻⁵ to 10⁻⁴ per generation, an efficiency which is in the same neighborhood as has been described for experiments performed in the messier mutator background. The authors' analysis of a series of gene conversion events shows that transgene sequences extend a median distance of 500 bp on either side of the DSB. Although researchers interested in using this strategy will want to consult the paper for details regarding relative efficiency and desired lengths of homologous sequences, the strategy is clearly appropriate for introducing point mutations, knockouts, or GFP knockins. Of course, an elegant approach means little in the absence of relevant reagents. Fortunately, there is an ongoing European collaboration to develop an extensive library of Mos1 insertions. Interested parties will find progress catalogued by Wormbase annotations and may visit elegans.imbb.forth.gr/nemagenetag for further information. –ND

-Robert and Bessereau. 2006. Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks. The EMBO Journal [Epub ahead of print, December 7, 2006].

Accelerating Transfection

There's no question that lipofection is a fast transfection protocol from a user standpoint, but from a molecular perspective, true speed means bombardment via gene gun. Classically, particle-mediated delivery involves linking DNA to gold microparticle carriers. This technology has a strong advantage in that it isn't limited to particular cell types, but it does have limitations. The carrier particles may disturb the cells they end up within, and the particle conjugation process can be inappropriate for use with proteins. For a group of Taiwanese researchers, the solution was obvious: ditch the particles and try “molecular bombardment” instead, in essence, a strategy in which the payload becomes the missile. In a new report appearing in Experimental Cell Research, (Lian et al) show successful penetration of a variety of cell types with naked (carrier-free) molecules of varying types. To convince themselves that molecular bombardment works, the authors first demonstrated successful transfer of dyes (Hoechst and Lucifer yellow) and different sized dextrans into CHO cells. Lian et al. then tested direct introduction of plasma DNA, achieving transfection efficiencies of 35% (CHO cells) and 10% (Hep G2, a differentiated cell type). More impressively, the technique could also be used for DNA delivery into neuronal cells in goldfish retina explants, a target that is generally very difficult to transfect. The most critical tests concerned the use of molecular bombardment for protein delivery. The authors present data showing that rhodamine-labeled actin and tubulin could be successfully introduced into both cultured (CHO) or primary (fish keratocyte) cells, as shown by incorporation of the labeled proteins in the cytoskeleton. Notably, molecular bombardment is not strictly limited to molecules, as the authors also demonstrate successful penetration of E. coli into Hep G2 cells. All in all, Lian et al. make a strong case that molecular bombardment represents an attractive transfection method. Cell survival is in excess of 80%, and the amount of protein required is less than that needed for electroporation or delivery by shockwave or ultrasound. Although the exact mechanism by which the method succeeds remains speculative, the success of these molecular bombardment experiments shows that gene guns can effectively use a much wider range of ammunition than previously thought. –ND

-Lian et al. 2007. Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles. Experimental Cell Research 313:53-64.