RESUMO
The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.
Assuntos
Bancos de Espécimes Biológicos , Genômica/métodos , Haploidia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Animais , Vasos Sanguíneos/citologia , Linhagem da Célula/genética , Resfriado Comum/genética , Resfriado Comum/virologia , Genes Essenciais/genética , Testes Genéticos , Células HEK293 , Homozigoto , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Neovascularização Fisiológica/genética , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Independentes de Cálcio/metabolismo , Rhinovirus/patogenicidadeRESUMO
Lactoferrin (Lf) is a multifunctional glycoprotein. Due to its anti-inflammatory and anti-cancer properties and the resulting therapeutical potential, lactoferrin is at present focus of a variety of research areas. The regulation of cell growth represents one of the prominent performances of lactoferrin. In this study we found lactoferrin to inhibit proliferation of the human epithelial cancer cell line HeLa. The extent of this growth inhibition was comparable to the one induced by the transforming-growth-factor-beta-1 (TGFbeta1). In contrast to other cell lines where lactoferrin stimulates growth, lactoferrin failed to activate the MAP kinases ERK1/2 or p38 in HeLa cells. However, by immunocytochemistry and cell fractionation experiments, we found that lactoferrin is capable of activating the TGFbeta/Smad-2 pathway. The nuclear accumulation of Smad-2 induced by Lf was comparable in magnitude to the one induced by TGFbeta1. We therefore conclude that the canonical TGFbeta1 pathway is a feasible route for lactoferrin to transduce its antiproliferative effect in HeLa cells, when MAPkinase activation is absent.
