Sparking new frontiers: using in vivo electroporation for genetic manipulations

In vivo electroporation is a fascinating new approach by which gene expression, regulation, and function can be studied in developmental systems. This technique offers new opportunities for manipulations in animal models that lack genetic approaches, including avians. Furthermore, this approach is applicable to other embryo populations including mice, ascidians, zebrafish, Xenopus, and Drosophila. In this review, we discuss technical aspects of in vivo electroporation, review recent studies where this approach has been utilized successfully, and identify future directions.     

Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR

AIMS: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. METHODS AND RESULTS: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. CONCLUSIONS: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.     

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.     

Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus

Bioprocess and Biosystems Engineering - Bioreactors at the microliter scale offer a promising approach to accelerate bioprocess development. Advantages of such microbioreactors include a reduction...     

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.