Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.     

Distribution and Numbers of Pygmies in Central African Forests

Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. However, their numbers and actual distribution is not known precisely. Here, we undertake this task by using locational data and population sizes for an unprecedented number of known Pygmy camps and settlements (n = 654) in five of the nine countries where currently distributed. With these data we develop spatial distribution models based on the favourability function, which distinguish areas with favourable environmental conditions from those less suitable for Pygmy presence. Highly favourable areas were significantly explained by presence of tropical forests, and by lower human pressure variables. For documented Pygmy settlements, we use the relationship between observed population sizes and predicted favourability values to estimate the total Pygmy population throughout Central Africa. We estimate that around 920,000 Pygmies (over 60% in DRC) is possible within favourable forest areas in Central Africa. We argue that fragmentation of the existing Pygmy populations, alongside pressure from extractive industries and sometimes conflict with conservation areas, endanger their future. There is an urgent need to inform policies that can mitigate against future external threats to these indigenous peoples’ culture and lifestyles.     

Detection of a long acting endogenous opioid in blood and small intestine.

The blood of guinea-pigs and certain other species was found to contain two substances with opiate-like activity. These two substances could be separated by thin layer chromatography in a variety of solvent systems, which enabled them to be categorised as either fast or slow moving material. Although both substances caused a naloxone-antagonisable inhibition of the twitch tension of the electrically-stimulated myenteric plexus-longitudinal muscle strip from the guinea-pig ileum, the fast moving material differed in that its effect could only be reversed by many repeated washings of the preparation. Both fast and slow moving material were found to be 30 times less potent on the isolated mouse vas deferens than on the guinea-pig ileum preparation. The inhibiting effects of these opioids were not altered by incubation with either trypsin or pronase. An opioid was also detected in the fluid bathing strips of the guinea-pig ileum preparation. This opioid had similar properties to the fast moving material isolated from blood. The release of this material from strips of the guinea-pig ileum was not enhanced by electrical stimulation of the preparation.     

Cell cycle-dependent reactivity with the monoclonal antibody Ki-67 during myeloid cell differentiation

The specificity and sensitivity of the monoclonal antibody Ki-67 in identifying proliferating cell compartments was tested with the human promyelocytic leukemia cell line HL-60 using multi-parameter flow cytometry. While correlated measurements of DNA content and Ki-67 immunofluorescence indicated that the antigen was present in all phases of the cell cycle, reactivity with the antibody was highest in proliferating S and G2+M cells. The analysis of the BrdU content of cells sorted on the basis of reactivity with Ki-67 showed a correlation between Ki-67 reactivity and BrdU uptake. In HL-60 cells induced to differentiate with dimethyl sulfoxide (DMSO), the loss of reactivity with Ki-67 paralleled the exit of cells from the cell cycle. This was not observed in DMSO-resistant HL-60 cells. These results validate the usefulness of the Ki-67 antibody for determining the proliferative stage of mammalian cells in culture.     

Dispersal and field progeny production of Trichogramma species released in an olive orchard in Egypt

Dispersal ability and field progeny production of augmentative released biological control agents depend on ecological adaptations of the particular species or strains used. Four species of the egg parasitoid genus Trichogramma were compared aiming to select suitable candidates for control of lepidopteran olive pests. Three of them (T. bourarachae Pintureau and Babault, T. cordubensis Vargas and Cabello, T. euproctidis Girault) had been previously collected from olive groves, whereas the commercially available strain used (T. evanescens Westwood) was originally isolated from sugarcane fields. During five consecutive field releases in an olive orchard near Cairo, dispersal and/or progeny production of these species was monitored using sentinel eggs placed at different heights in the release tree canopies as well as in neighboring trees (“distance effect”). The cardinal direction of dispersal was random for T. euproctidis and T. evanescens. Significant higher parasitism occurred on sentinel eggs placed on the middle part of tree canopy and highest parasitism was observed in trees where wasps had been released. Field progeny production was highest for T. bourarachae, followed by T. euproctidis and T. cordubensis. T. evanescens propagated less under field conditions. Inter-tree dispersal of all species except T. bourarachae was limited and, for biological control, releasing material should therefore be distributed on each olive tree, preferably also at different levels of the canopy.