Transposon mutagenesis of the mouse germline.

Sleeping Beauty is a synthetic "cut-and-paste" transposon of the Tc1/mariner class. The Sleeping Beauty transposase (SB) was constructed on the basis of a consensus sequence obtained from an alignment of 12 remnant elements cloned from the genomes of eight different fish species. Transposition of Sleeping Beauty elements has been observed in cultured cells, hepatocytes of adult mice, one-cell mouse embryos, and the germline of mice. SB has potential as a random germline insertional mutagen useful for in vivo gene trapping in mice. Previous work in our lab has demonstrated transposition in the male germline of mice and transmission of novel inserted transposons in offspring. To determine sequence preferences and mutagenicity of SB-mediated transposition, we cloned and analyzed 44 gene-trap transposon insertion sites from a panel of 30 mice. The distribution and sequence content flanking these cloned insertion sites was compared to 44 mock insertion sites randomly selected from the genome. We find that germline SB transposon insertion sites are AT-rich and the sequence ANNTANNT is favored compared to other TA dinucleotides. Local transposition occurs with insertions closely linked to the donor site roughly one-third of the time. We find that approximately 27% of the transposon insertions are in transcription units. Finally, we characterize an embryonic lethal mutation caused by endogenous splicing disruption in mice carrying a particular intron-inserted gene-trap transposon.

Mobile measurements and regression modeling of the spatial particulate matter variability in an urban area.

During 5 different periods between summer 2009 and spring 2011, mobile measurements were carried out in the city of Aachen, Germany, in order to capture the spatial variability of particulate matter concentrations in urban and suburban environments. Results show a large spatial variability on a scale of tens of meters, mainly depending on traffic density and building structure. Spatial coefficients of variation exhibit larger spatial variability for PM(10) than for PM(2.5) and larger variability in traffic influenced inner city environments than in suburban areas. Based on the results of an extensive campaign, a regression model is developed for the prediction of PM(10) and PM(2.5) distributions over the city area. The three predictors for the regression model are an exponential PM concentration profile simulated on the basis of PM(10) and PM(2.5) traffic emissions, building density and green area density within radii of 50 m and 100 m. The model shows good agreement between measured and modeled PM levels during the campaign used for the model training with R(2) values of 0.79 and 0.65, RMSE of 1.9 µg/m(3) and 1 µg/m(3) for PM(10) and PM(2.5), respectively. The model is further validated using data from the remaining measurement campaigns and modeling of PM levels at monitoring sites that were not used for the training of the regression model. For the total number of 59 monitoring sites, the regression model shows R(2) values of 0.77 (PM(10)) and 0.61 (PM(2.5)) with RMSE of 2.3 µg/m(3) and 1.2 µg/m(3). The modeled concentrations are generally in better accordance with measured concentrations for PM(10) than for PM(2.5) concentrations. We attribute this to higher spatial homogeneity of PM(2.5) levels compared to coarse particles. Inner city PM levels at traffic influenced sites are better reproduced by the model than suburban concentrations which exhibit the smallest spatial variability.

Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system.

Hemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain-deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.

Mammalian germ-line transgenesis by transposition.

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.