Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.     

A Genetic Analysis of Mancozeb Resistance in Typhlodromus Pyri (Acari: Phytoseiidae)

A field population of Typhlodromus pyri (Acari: Phytoseiidae) tolerant to mancozeb was selected in the laboratory. After 10 mancozeb selections the LC₅₀ value for mancozeb was 73 times higher in the selected-10 strain compared to the standard susceptible strain. A genetic analysis using reciprocal crosses and backcrosses of female F₁ progeny found no maternal effect. Resistance in the selected-10 strain was codominant in expression, dominance value was about −0.1. Backcrosses between F₁ females and the susceptible strain indicate that the resistance to mancozeb could be principally conferred by a predominant gene, but additional factors would also be involved.     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

Correlation between faecal microbial community structure and cholesterol-to-coprostanol conversion in the human gut

Intensity of the cholesterol-to-coprostanol conversion in the intestine, as assessed by the coprostanol-to-cholesterol ratio in faeces, was found highly variable among 15 human volunteers, ranging from absent to almost complete cholesterol conversion. The number of coprostanoligenic bacteria in the same faecal samples, as estimated by the most probable number method, was found to be less than 10(6) cellsg-1 of fresh stools in the low-to-inefficient converters and at least 10(8) cellsg-1 of fresh stools in the highest converters, indicating that the population level of cultivable faecal coprostanoligenic bacteria correlated with the intensity of cholesterol-to-coprostanol conversion in the human gut. Microbial communities of the samples were profiled by temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNA gene amplicons. Dendrogram analysis of the TTGE profiles using the Pearson product moment correlation coefficient and a unweighted pair group method with arithmetic averages (UPGMA) algorithm clearly separated banding patterns from low-to-inefficient and high converters in two different clusters suggesting a relationship between TTGE profiles and coprostanoligenic activity. Principal components analysis further demonstrated that a large subset of bands rather than some individual bands contributed to this clustering.