El ratolí com a sistema model en biologia

Des de l'establiment de les primeres soques de laboratori al començament del segle xx, el ratolí s'ha convertit en una eina fonamental en la recerca en biologia. Múltiples models experimentals han permès ampliar el nostre coneixement en camps com l'embriogènesi, la fisiologia o la biomedicina, entre d'altres. El desenvolupament de les tecnologies de modificació genètica ha consolidat definitivament el ratolí com el mamífer d'elecció a l'hora de caracteritzar patrons d'expressió, funcions i interaccions entre gens, així com en la modelització de malalties humanes i l'assaig de nous fàrmacs. Aquest capítol pretén oferir una visió general de les àmplies possibilitats que es deriven de l'ús del ratolí en el laboratori i inclou exemples que demostren per què és un element clau en la recerca en biologia actualment.The mouse as a model system in biology. Since the beginning of the 20th century, when the first laboratory strains were developed, the mouse has become a central tool in biological research. Many experimental models have allowed the broadening of our knowledge in scientific fields such as embryogenesis, physiology or biomedicine. Development of genetic modification techniques has definitely established the mouse as the mammal organism of choice for the characterization of gene expression patterns, functions and interactions among genes as well as for the modeling of human diseases and drug testing. This chapter wants to offer a general view on the possibilities derived from the use of the mouse in the laboratory, and includes examples showing why it has become a key element in biological research

A genome editing approach to study cancer stem cells in human tumors

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors

Zonation of Ribosomal DNA Transcription Defines a Stem Cell Hierarchy in Colorectal Cancer

Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5− tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins

Genealogies. Rescatar penyores

[ca] Com a «penyores»... coneixem aquells objectes que es deixen en poder d’algú, com a prova de retorn o com a promesa de record. Penyores són també aquells estris oblidats en espais en desús, peces descartades, de manera conscient o involuntària —descuidar-se, fugir. Els espais que ens envolten, on diàriament vivim i treballem, acumulen artefactes carregats de vivències, històries i faules de qui abans els ha emprat. Narren èxits, fracassos, formes de viure; si més no, la màgia de la quotidianitat. Independentment de l’estadi vital on s’estigui —joventut o senectut—, s’entén la importància i el potencial de reutilitzar el que s’ha llegat: per acostar-se a la memòria, rellegir-la, fer-ne crida, furgar-hi, reivindicar i, siguin els que siguin, propiciar canvis a situacions actuals. A «Genealogies. Rescatar penyores», artistes novells, artistes convidats i professorat de la Facultat de Belles Arts de la Universitat de Barcelona, amb la complicitat del públic assistent, miren d’aportar noves visions sobre els objectes i els temps pignorats que contenen, com a punt de partida per encetar un diàleg narratiu de tint feminista, econòmic, de cura, de sostenibilitat emocional

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression.

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.MF and EC were supported by respective FPI fellowships from the Ministerio de Economía y Competitividad. AMS held an AECC postdoctoral fellowship. AD-V was supported in part by a contract PTC2011-1091 from Ministerio de Economía y Competitividad. This work was supported by grants from the Ministerio de Economía y Competitividad/n(SAF2011/23638) to MAP; and an ERC grant to EB

Iro/IRX transcription factors negatively regulate Dpp/TGF-β pathway activity during intestinal tumorigenesis

© 2014 The Authors. Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal coactivation of these pathways in the adult Drosophila midgut induces >tumor-like> overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-β acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-β. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-β. In addition, Irx5 expression confers a growth advantage in the presence of TGF-β, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-β activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-β, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-β pathway components.O.M. is a recipient of a FPU fellowship from the MEC, and F.M.B holds an IRB/PhD La Caixa fellowship. This work was supported by the MICINN (BFU2010-16016 and BFU2011-23479) to A.C.Peer Reviewe