The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem URL.The BACFinder clone search and oligo design tool is available online at http://www.mitocheck.org/cgi-bin/BACfinder.Database accession codes. The ChIP/chip data has been submitted to the Gene Expression Omnibus database with accession number GSE10845. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/naturemethods/. Europe PMC Funders GroupAuthor Manuscript Nat Methods. Author manuscript; available in PMC 2010 May 17. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.At a time when the 'thousand-dollar genome' seems a realistic goal for the near future, methods for dissecting the functions of the encoded genetic information lag far behind the genome sequence, both in throughput and in quality of the produced data. Genome sequencing and subsequent bioinformatics analysis have made it possible to study the function of genes in mammalian tissue culture cells using systematic reverse-genetic approaches1-3 and have radically improved researchers' ability to identify human disease genes. Such studies typically identify single genes, whose biological function has often not yet been described. In order to place the proteins these genes encode in pathways, these studies must be followed by detailed molecular-level analysis, of which the most powerful types are protein localization and protein-protein interaction. The power of protein localization and protein-protein interaction studies can be seen from the genome-wide application of GFP localization and tandem affinity tag-based complex purification in the yeast Saccharomyces cerevisiae, which has produced a comprehensive picture of the core proteome of a simple, well-studied model system4-8. The key advantage of yeast for these studies was their efficient intrinsic homologous recombination, which allowed the same tagcoding sequence to be introduced at the endogenous locus of nearly every gene of the genome. The tagged proteins were then systematically analyzed through standardized, generic, tag-based assays.To transfer this approach to mammali...

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Abstract. This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome composite probes. The colocalization of a 4,6-diamidino-2-phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D-reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the two chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/ TIE2 interaction during sepsis is a potential therapeutic target.