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1.
Nature ; 474(7351): 337-42, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21677750

RESUMO

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.


Assuntos
Deleção de Genes , Técnicas de Inativação de Genes/métodos , Genes/genética , Estudos de Associação Genética/métodos , Genoma/genética , Camundongos Knockout/genética , Alelos , Animais , Biologia Computacional , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genes Letais/genética , Vetores Genéticos/genética , Genômica , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional/métodos , Fenótipo , Reação em Cadeia da Polimerase , Ratos
2.
Front Cell Infect Microbiol ; 13: 1287355, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38173794

RESUMO

Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.


Assuntos
Malária Falciparum , Malária , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Malária/parasitologia , Eritrócitos/parasitologia , Células-Tronco
3.
Mamm Genome ; 23(9-10): 580-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22968824

RESUMO

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.


Assuntos
Camundongos Knockout/genética , Animais , Internacionalidade , Internet , Camundongos
4.
PLoS Genet ; 4(3): e1000016, 2008 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-18369441

RESUMO

The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human beta-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced.


Assuntos
Globinas/genética , Região de Controle de Locus Gênico , Família Multigênica , Animais , Sequência de Bases , Cromatina/genética , Primers do DNA/genética , Feminino , Feto/metabolismo , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Hibridização in Situ Fluorescente , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez , Transcrição Gênica
5.
EMBO J ; 21(17): 4612-20, 2002 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-12198163

RESUMO

While an important role for the POU domain transcription factor Oct-6 in the developing peripheral nerve has been well established, studies into its exact role in nerve development and regeneration have been hampered by the high mortality rate of newborn Oct-6 mutant animals. In this study we have generated a Schwann cell-specific Oct-6 allele through deletion of the Schwann cell-specific enhancer element (SCE) in the Oct-6 locus. Analysis of mice homozygous for this allele (deltaSCE allele) reveals that rate-limiting levels of Oct-6 in Schwann cells are dependent on the SCE and that this element does not contribute to Oct-6 regulation in other cell types. We demonstrate a Schwann cell autonomous function for Oct-6 during nerve development as well as in regenerating nerve. Additionally, we show that Krox-20, an important regulatory target of Oct-6 in Schwann cells, is activated, with delayed kinetics, through an Oct-6-independent mechanism in these mice.


Assuntos
Elementos Facilitadores Genéticos , Regeneração Nervosa/fisiologia , Nervos Periféricos/crescimento & desenvolvimento , Células de Schwann/fisiologia , Fatores de Transcrição/fisiologia , Alelos , Animais , Linhagem da Célula , Quimera , Cruzamentos Genéticos , Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce , Elementos Facilitadores Genéticos/genética , Feminino , Marcação de Genes , Masculino , Camundongos , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fator 6 de Transcrição de Octâmero , Especificidade de Órgãos , Nervos Periféricos/anatomia & histologia , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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