摘要 :
More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by i...
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More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative onco-genes such as EGFR, KRAS. MYC. BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1. and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.
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To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured...
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To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured embryonic stem cells and freshly isolated cumulus cells. Direct comparison of gene expression profiles of more than 10, 000 genes showed that for both donor cell types ≈4% of the expressed genes in the NT placentas differed dramatically in expression levels from those in controls and that the majority of abnormally expressed genes were common to both types of clones. Importantly, however, the expression of a smaller set of genes differed between the embryonic stem cell- and cumulus cell-derived dones. The livers of the cloned mice also showed abnormal gene expression, although to a lesser extent, and with a different set of affected genes, than seen in the placentas. Our results demonstrate frequent abnormal gene expression in clones, in which most expression abnormalities appear common to the NT procedure whereas others appear to reflect the particular donor nucleus.
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We describe a strategy to obtain highly enriched long-term repopulating (LTR) hematopoietic stem cells (HSCs) from bone marrow side-population (SP) cells by using a transgenic reporter gene driven by a stem cell enhancer. To analy...
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We describe a strategy to obtain highly enriched long-term repopulating (LTR) hematopoietic stem cells (HSCs) from bone marrow side-population (SP) cells by using a transgenic reporter gene driven by a stem cell enhancer. To analyze the gene-expression profile of the rare HSC population, we developed an amplification protocol termed "constant-ratio PCR," in which sample and control cDNAs are amplified in the same PCR. This protocol allowed us to identify genes differentially expressed in the enriched LTR-HSC population by oligonucleotide microarray analysis using as little as 1 ng of total RNA. Endoglin, an ancillary transforming growth factor β receptor, was differentially expressed by the enriched HSCs. Importantly, endoglin-positive cells, which account for 20% of total SP cells, contain all the LTR-HSC activity within bone marrow SP. Our results demonstrate that endoglin, which plays important roles in angiogenesis and hematopoiesis, is a functional marker that defines LTR HSCs. Our overall strategy may be applicable for the identification of markers for other tissue-specific stem cells.
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摘要 :
The genetic architecture of complex traits underlying physiology and disease in most organisms remains elusive. We still know little about the number of genes that underlie these traits, the magnitude of their effects, or the exte...
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The genetic architecture of complex traits underlying physiology and disease in most organisms remains elusive. We still know little about the number of genes that underlie these traits, the magnitude of their effects, or the extent to which they interact. Chromosome substitution strains (CSSs) enable statistically powerful studies based on testing engineered inbred strains that have single, unique, and nonoverlapping genetic differences, thereby providing measures of phenotypic effects that are attributable to individual chromosomes. Here, we report a study of phenotypic effects and gene interactions for 90 blood, bone, and metabolic traits in a mouse CSS panel and 54 traits in a rat CSS panel. Two key observations emerge about the genetic architecture of these traits. First, the traits tend to be highly polygenic: across the genome, many individual chromosome substitutions each had significant phenotypic effects and, within each of the chromosomes studied, multiple distinct loci were found. Second, strong epistasis was found among the individual chromosomes. Specifically, individual chromosome substitutions often conferred surprisingly large effects (often a substantial fraction of the entire phenotypic difference between the parental strains), with the result that the sum of these individual effects often dramatically exceeded the difference between the parental strains. We suggest that strong, pervasive epistasis may reflect the presence of several phenotypically-buff-ered physiological states. These results have implications for identification of complex trait genes, developmental and physiological studies of phenotypic variation, and opportunities to engineer phenotypic outcomes in complex biological systems.
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The international Human Genome Project (HGP) and Celera Genomics published articles last year on the sequence of the human genome (1, 2). In a recent article (3), we analyzed aspects of the Celera article. We noted that the articl...
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The international Human Genome Project (HGP) and Celera Genomics published articles last year on the sequence of the human genome (1, 2). In a recent article (3), we analyzed aspects of the Celera article. We noted that the article did not report an assembly of Celera's own data but rather reported only joint assemblies based on a data set that included the assembled genome sequence of the HGP. Approximately 60% of the underlying sequence data and 100% of the mapping data used in Celera's analysis came from the HGP, and the HGP genome assembly itself contained 90% of the euchromatic sequence of the human genome. We also noted that Celera used various approaches for using the HGP data (referred to as perfect tiling, gap filling, and compartmentalized assembly; see Fig. 1) that implicitly preserved much of the HGP assembly information. We concluded that Celera's assemblies made extensive and inextricable use of the HGP genome information and thus were not an independent assembly of the human genome.
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