http://repub.eur.nl/res/aut/3512/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33840/ 2011-09-01T00:00:00Z Background: In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. Design and Methods: To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. Results: We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRASlike suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. Conclusions: We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the downregulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins. http://repub.eur.nl/res/pub/23951/ 2010-01-01T00:00:00Z Germ line gene transposition technology has been used to generate "libraries" of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular "disease" phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes. Copyright http://repub.eur.nl/res/pub/14099/ 2006-10-10T00:00:00Z We have generated transgenic mice containing hybrid llama/human antibody loci that contain two llama variable regions and the human D, J, and Cmu and/or Cgamma constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigen-specific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future. http://repub.eur.nl/res/pub/10237/ 2003-01-01T00:00:00Z The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation. http://repub.eur.nl/res/pub/2552/ 1998-01-01T00:00:00Z Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo. http://repub.eur.nl/res/pub/2547/ 1997-10-01T00:00:00Z The cellular retinoic acid binding protein type I (CRABP-I) shows a highly specific expression pattern during mouse embryonic development. The tissues that express CRABP-I, i.e. the central nervous system (CNS), neural crest, branchial arches, limb bud and frontonasal mass, coincide with those that are most sensitive to unphysiological retinoic acid (RA) concentrations. We have investigated the transcriptional elements that are responsible for the spatiotemporal regulation of CRABP-I expression in the mouse embryo. We show here that a 16 kb fragment harbours all the elements needed for the correct spatiotemporal expression pattern. Upon further dissection of this fragment we have found that expression in the CNS is driven by elements in the upstream region of the gene, while expression in mesenchymal and neural crest tissue is regulated via element(s) located downstream of exon II of the gene. Two distinct fragments in the upstream region are required for expression in the CNS, as neither of these fragments alone is able to drive correct expression of a reporter gene in transgenic mice. DNAseI footprinting analysis of the two upstream fragments revealed the presence of a number of protected elements. One of these regulatory elements has the hallmarks of an RA response element, suggesting that CRABP-I expression in neural tissue can be directly modulated by RA via the RARs/RXRs.