Mammalian embryo co-culture: trials and tribulations of a misunderstood method

Embryo-somatic cell co-culture was devised over 40 years ago in an attempt to improve the development and viability of mammalian preimplantation embryos generated and cultured in vitro. While initial endeavours were successful in this respect, other studies soon highlighted a number of significant long-term detrimental impacts of this approach. Surprisingly little is known about the mechanisms underlying the beneficial effects of co-culture, although the production of embryotrophic compounds, modulation of nutrient profile, protection against culture-induced stress and/or toxin clearance are all contenders. The extent to which the inadvertent exposure of embryos to serum accounts for many of these effects remains open to question. Although the popularity of somatic cell co-culture has recently declined in favour of the use of sequential media due to concerns associated with its risk of disease transmission and long-term sequelae, we argue that complete dismissal of this technique is ill advised, given that our limited understanding of basic somatic cell interactions has prevented us from fully exploiting its potential. In this respect, there is some merit in focussing future research strategies based on reconstructed maternal tract tissue. Although the use of co-culture in clinical practice is unacceptable and its implementation in domestic species for commercial purposes should be viewed with diffidence, this technique can still provide a wealth of information on the development of novel, more physiological embryo in vitro culture systems. The proviso for acquiring such information is to gain a fuller understanding of the culture requirements/biochemistry of somatic cells and their interaction with the early conceptus.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

Gene therapy: progress and predictions

The first clinical gene delivery, which involved insertion of a marker gene into lymphocytes from cancer patients, was published 25 years ago. In this review, we describe progress since then in gene therapy. Patients with some inherited single-gene defects can now be treated with their own bone marrow stem cells that have been engineered with a viral vector carrying the missing gene. Patients with inherited retinopathies and haemophilia B can also be treated by local or systemic injection of viral vectors. There are also a number of promising gene therapy approaches for cancer and infectious disease. We predict that the next 25 years will see improvements in safety, efficacy and manufacture of gene delivery vectors and introduction of gene-editing technologies to the clinic. Gene delivery may also prove a cost-effective method for the delivery of biological medicines.     

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.     

Field note: successful establishment of a phytoremediation system at a petroleum hydrocarbon contaminated shallow aquifer: trends, trials, and tribulations

We report the establishment of a mixed hybrid poplar (Populus spp.) and willow (Salix spp.) phytoremediation system at a fuel-contaminated site. Several approaches were used to balance competing goals of cost-effectiveness yet successful tree establishment without artificial irrigation or trenching. Bare root and unrooted cuttings were installed using either: (1) 1.2 m deep holes excavated with an 8 cm diameter auger using a direct-push rig and backfilled with the excavated, in situ soil; (2) 1.2 m deep holes created with a 23 cm diameter auger attached to a Bobcat rig and backfilled with clean topsoil from offsite; and (3) shallow holes between 15-30 cm deep that were created with a 1.3 cm diameter rod and no backfill. Tree mortality from initial plantings indicated contaminated zones not quantified in prior site investigations and remedial actions. Aquifer heterogeneity, underground utilities, and prior remediation infrastructure hampered the ability of the site to support a traditional experimental design. Total stem length and mortality were measured for all planted trees and were incorporated into a geographic information system. Planting early in the growing season, augering a larger diameter hole, and backfilling with clean, uncontaminated topsoil was cost effective and allowed for greater tree cutting growth and survival.