BACKGROUNDMutations in the β-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach ...to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.3–60% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported. METHODSZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses. RESULTSAlthough the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 1–2% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease. CONCLUSIONSWith ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.
We describe a novel gene targeting strategy for the genetic analysis of essential genes in mammalian cells and its use to study the role of the cell cycle control gene CDC2 in human cells. A cell ...line (HT2-19) was generated in which endogenous CDC2 gene expression and cell viability depend on the presence of an inducer in the growth medium. In the absence of inducer, HT2-19 cells undergo extensive DNA rereplication and apoptosis. Rereplication is indicative of a role for human CDC2 in a control mechanism, previously undetected in mammalian cells, that prevents premature entry into S-phase.
Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by α - and γ -interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic ...clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5′ flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-α ,β . Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-γ as well as IFN-α . Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5′ side of a marker gene. Since the 6-16 gene does not normally respond to IFN-γ , the context of the ISRE must determine the specificity of the response.
Wheat grain protein concentration is an important determinant of wheat quality for human nutrition that is often overlooked in efforts to improve crop production. We tested and applied a ...32‐multi‐model ensemble to simulate global wheat yield and quality in a changing climate. Potential benefits of elevated atmospheric CO2 concentration by 2050 on global wheat grain and protein yield are likely to be negated by impacts from rising temperature and changes in rainfall, but with considerable disparities between regions. Grain and protein yields are expected to be lower and more variable in most low‐rainfall regions, with nitrogen availability limiting growth stimulus from elevated CO2. Introducing genotypes adapted to warmer temperatures (and also considering changes in CO2 and rainfall) could boost global wheat yield by 7% and protein yield by 2%, but grain protein concentration would be reduced by −1.1 percentage points, representing a relative change of −8.6%. Climate change adaptations that benefit grain yield are not always positive for grain quality, putting additional pressure on global wheat production.
Potential benefits of elevated atmospheric CO2 concentration by 2050 on global wheat grain and protein yield are likely to be negated by impacts from rising temperature and changes in rainfall, but with considerable disparities between regions. Grain and protein yields are expected to be lower and more variable in most low‐rainfall regions, with nitrogen availability limiting growth stimulus from elevated CO2. Introducing genotypes adapted to warmer temperatures could boost global wheat yield by 7% and protein yield by 2%, but grain protein concentration would be reduced by −1.1% points, representing a relative change of −8.6%.
DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome ...structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.
The renal function is often affected in asphyxiated newborn infants. The pharmacokinetics of drugs like aminoglycosides eliminated through the kidneys may be impaired and require a different than ...usual dosage regimen. A decrease in body temperature is associated with a decrease in glomerular filtration rate and may, therefore, impair the elimination of aminoglycosides. When hypothermia is applied as neuronal rescue therapy after birth asphyxia, the pharmacokinetics of kidney-eliminated drugs may be impaired even more. We used our well-established global hypoxia-asphyxia newborn pig model to evaluate the effect of mild hypothermia after hypoxia-ischemia on gentamicin pharmacokinetics. Newborn pigs underwent global hypoxia-ischemia followed by normothermia (39 degrees C) for 72 h (n = 8) or mild hypothermia (35 degrees C) for 24 h followed by normothermia (39 degrees C) for 48 h (n = 8). Gentamicin pharmacokinetics was studied after three gentamicin doses: before hypoxia-ischemia, after hypoxia-ischemia during mild hypothermia or normothermia, and during normothermia 48 h after the first dose. The gentamicin pharmacokinetics variables were calculated using a SAAM II program. Hypoxia-ischemia altered renal function and gentamicin pharmacokinetics. The gentamicin clearance correlated with the creatinine plasma concentration (r = 0.89) and with the kidney pathology score (r = 0.55). There was no significant difference in gentamicin pharmacokinetics at 35 and 39 degrees C in newborn pigs after hypoxia-ischemia. The gentamicin pharmacokinetics variables were not different in the hypothermic or normothermic pigs after all three studied doses. Mild hypothermia for 24 h after hypoxia-ischemia does not affect gentamicin pharmacokinetics.
The full power of gene modification in higher eukaryotes, including its potential to correct disease-causing mutations, has so far been limited by its low efficiency. Now that power looks set to be ...released through the use of customized endonuclease "hands," DNA-binding zinc fingers linked to nuclease "thumbs," capable of grasping and cutting any chosen genomic target locus. Once cleaved, the target locus is efficiently repaired by endogenous mechanisms, using an exogenous DNA template that carries the chosen modification. These helping hands are likely to touch many areas of biological and clinical endeavor.
Vaccination has transformed public health, most notably including the eradication of smallpox. Despite its profound historical importance, little is known of the origins and diversity of the viruses ...used in smallpox vaccination. Prior to the twentieth century, the method, source and origin of smallpox vaccinations remained unstandardised and opaque. We reconstruct and analyse viral vaccine genomes associated with smallpox vaccination from historical artefacts. Significantly, we recover viral molecules through non-destructive sampling of historical materials lacking signs of biological residues. We use the authenticated ancient genomes to reveal the evolutionary relationships of smallpox vaccination viruses within the poxviruses as a whole.
Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human ...pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.
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•Genomic, epigenomic, and transcriptomic characterization of sporadic glioma in dogs•Somatic alterations in canine glioma converge with human glioma drivers•Canine glioma resemble pediatric human glioma by mutation rate and DNA methylation•Microenvironment similarity between canine and human pediatric and adult glioma
Amin et al. characterize the molecular landscape of canine gliomas and compare it with that of human pediatric and adult gliomas, revealing high similarity between human pediatric and canine gliomas. The cross-species analysis identifies conserved glioma drivers and aneuploidy as a hallmark of high-grade disease.