Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ～1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.     

Pharmacogenetics meets metabolomics: discovery of tryptophan as a new endogenous OCT2 substrate related to metformin disposition

Genetic polymorphisms of the organic cation transporter 2 (OCT2), encoded by SLC22A2, have been investigated in association with metformin disposition. A functional decrease in transport function has been shown to be associated with the OCT2 variants. Using metabolomics, our study aims at a comprehensive monitoring of primary metabolite changes in order to understand biochemical alteration associated with OCT2 polymorphisms and discovery of potential endogenous metabolites related to the genetic variation of OCT2. Using GC-TOF MS based metabolite profiling, clear clustering of samples was observed in Partial Least Square Discriminant Analysis, showing that metabolic profiles were linked to the genetic variants of OCT2. Tryptophan and uridine presented the most significant alteration in SLC22A2-808TT homozygous and the SLC22A2-808G>T heterozygous variants relative to the reference. Particularly tryptophan showed gene-dose effects of transporter activity according to OCT2 genotypes and the greatest linear association with the pharmacokinetic parameters (Cl(renal), Cl(sec), Cl/F/kg, and Vd/F/kg) of metformin. An inhibition assay demonstrated the inhibitory effect of tryptophan on the uptake of 1-methyl-4-phenyl pyrinidium in a concentration dependent manner and subsequent uptake experiment revealed differential tryptophan-uptake rate in the oocytes expressing OCT2 reference and variant (808G>T). Our results collectively indicate tryptophan can serve as one of the endogenous substrate for the OCT2 as well as a biomarker candidate indicating the variability of the transport activity of OCT2.     

A multicenter study confirms CD226 gene association with systemic sclerosis-related pulmonary fibrosis

Idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune diseases that are characterized by symmetrical skeletal muscle weakness and muscle inflammation with no known cause. Like other autoimmune diseases, IIMs are treated with either glucocorticoids or immunosuppressive drugs. However, many patients with an IIM are frequently resistant to immunosuppressive treatments, and there is compelling evidence to indicate that not only adaptive immune but also several non-immune mechanisms play a role in the pathogenesis of these disorders. Here, we focus on some of the evidence related to pathologic mechanisms, such as the innate immune response, endoplasmic reticulum stress, non-immune consequences of MHC class I overexpression, metabolic disturbances, and hypoxia. These mechanisms may explain how IIM-related pathologic processes can continue even in the face of immunosuppressive therapies. These data indicate that therapeutic strategies in IIMs should be directed at both immune and non-immune mechanisms of muscle damage.     

Transport of beta-globin mRNA from nuclei of murine Friend erythroleukemia cells. Reversible inhibition of transport by the oxidizing sulfhydryl reagent o-iodosobenzoate

An in vitro assay system for analysis of beta-globin mRNA transport is described. Nuclei isolated from murine Friend erythroleukemia cells induced to synthesize globin mRNA, were incubated in micro-assays. By electrophoresis and hybridization analysis, released 9-S beta-globin mRNA was shown to be undegraded. After direct blotting, the released mRNA was quantified by hybridization with a labeled plasmid containing a beta-globin DNA restriction fragment. The inducibility of beta-globin mRNA transport corresponded to that previously reported for the release of rapidly labeled RNA in other assay systems. In contrast to the ineffectiveness of high concentrations of the sulfhydryl reagent iodoacetate, low concentrations of the oxidizing sulfhydryl reagent, o-iodosobenzoate, inhibited the release of beta-globin mRNA from nuclei of erythroleukemia cells, as well as the release of rapidly labeled RNA from rat liver nuclei. The inhibitory effect of the oxidizing agent on beta-globin mRNA transport could be reversed by postincubation of the nuclei with the reducing agent, dithiothreitol. The potential role of disulfide bond formation on RNA transport is discussed.     

Phylogenetic relationships among prokaryotic and eukaryotic catalases

Seventy-four catalase protein sequences, including 29 bacterial, 8 fungal, 7 animal, and 30 plant sequences, were compiled, and 70 were used for phylogenetic reconstruction. The core of the resulting tree revealed unique, separate groups of plant and animal catalases, two groups of fungal catalases, and three groups of bacterial catalases. The only overlap of kingdoms occurred within one branch and involved fungal and bacterial large-subunit enzymes. The other fungal branch was closely linked to the group of animal enzymes. Group I bacterial catalases were more closely related to the plant enzymes and contained such diverse taxa as the Gram-positive Listeria seeligeri, Deinocococcus radiodurans, and gamma-proteobacteria. Group III bacterial sequences were more closely related to fungal and animal sequences and included enzymes from a broad range of bacteria including high- and low-GC Gram positives, proteobacteria, and a bacteroides species. Group II was composed of large-subunit catalases from diverse sources including Gram positives (low-GC Bacilli and high-GC Mycobacteria), proteobacteria, and species of the filamentous fungus Aspergillus. These data can be interpreted in terms of two gene duplication events that produced a minimum of three catalase gene family members that subsequently evolved in response to environmental demands. Horizontal gene transfer may have been responsible for the group II mixture of bacterial and fungal large-subunit catalases.      

The placenta releases branched-chain keto acids into the umbilical and uterine circulations in the pregnant sheep

There was net uptake of branched-chain keto acids by the fetus from the umbilical circulation. Mean fetal uptake of the 3 keto acids 2-keto isovalerate, 2-keto isocaproate and 2-keto methylvalerate was 1.8 mumol/min per kg of fetus. The concentrations in the umbilical vein for these keto acids were 10.9 +/- 3.8 microM (mean +/- SD: 2-keto isovalerate), 19.7 +/- 6.1 microM (2-keto isocaproate) and 14.8 +/- 5.3 microM (2-keto methylvalerate) respectively. The coefficients of extraction for the same keto acids were 17.2%, 16.8% and 11.9% respectively. Fetal uptakes (both mumol/min and mumol/min per kg fetus) were positively correlated with umbilical supply. There were concentration gradients across the placenta, with fetal concentration: maternal concentration ratios of 3.3 +/- 1.5 for 2-keto isovalerate, 2.1 +/- 0.8 for 2-keto isocaproate and 1.3 +/- 0.6 for 2-keto methylvalerate. The net release of 2-keto acids into the umbilical circulation may conserve the carbon skeleton of branched-chain amino acids for fetal metabolism and growth. In the uterine circulation there was not a consistent pattern of release from or uptake by the uteroplacental tissues. It is suggested that branched-chain keto acids may contribute to fetal growth or energy metabolism.     

Quality control for plant metabolomics: reporting MSI-compliant studies

The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum parameters for reporting metabolomics experiments, in order to validate metabolomic studies and to facilitate data exchange. The reporting parameters encompassed by MSI include the biological study design, sample preparation, data acquisition, data processing, data analysis and interpretation relative to the biological hypotheses being evaluated. Herein we exemplify how such metadata can be reported by using a small case study – the metabolite profiling by GC-TOF mass spectrometry of Arabidopsis thaliana leaves from a knockout allele of the gene At1g08510 in the Wassilewskija ecotype. Pitfalls in quality control are highlighted that can invalidate results even if MSI reporting standards are fulfilled, including reliable compound identification and integration of unknown metabolites. Standardized data processing methods are proposed for consistent data storage and dissemination via databases.     

The impact of the neisserial DNA uptake sequences on genome evolution and stability

Background  

Efficient natural transformation in Neisseria requires the presence of short DNA uptake sequences (DUSs). Doubts remain whether DUSs propagate by pure selfish molecular drive or are selected for 'safe sex' among conspecifics.