Jan Willem Voncken
Maastricht University Medical Center, Molecular Genetics, Faculty Member
Introduction of germ line mutations in mice via genetic engineering involves alterations of the structure and characteristics of genes. These alterations are mostly introduced via molecular genetic technology either in embryonal stem... more
Introduction of germ line mutations in mice via genetic engineering involves alterations of the structure and characteristics of genes. These alterations are mostly introduced via molecular genetic technology either in embryonal stem cells or in one-cell stage embryos. This chapter describes classic biotechnological methods used to generate mice from modified pre-implantation embryos.
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Transgenics are powerful mouse models to understand the biological functions of genes. This chapter gives a short overview of the requirements and considerations in designing a transgene. In addition, potential important choices that have... more
Transgenics are powerful mouse models to understand the biological functions of genes. This chapter gives a short overview of the requirements and considerations in designing a transgene. In addition, potential important choices that have to be made in advance for the successful designing and generating a transgenic mouse model are discussed. Methods for DNA purification for microinjection are also provided in this chapter.
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The nuclear envelope and the lamina define the nuclear periphery and are implicated in many nuclear processes including chromatin organization, transcription and DNA replication. Mutations in lamin A proteins, major components of the... more
The nuclear envelope and the lamina define the nuclear periphery and are implicated in many nuclear processes including chromatin organization, transcription and DNA replication. Mutations in lamin A proteins, major components of the lamina, interfere with these functions and cause a set of phenotypically diverse diseases referred to as laminopathies. The phenotypic diversity of laminopathies is thought to be the result of alterations in specific protein- and chromatin interactions due to lamin A mutations. Systematic identification of lamin A-protein and -chromatin interactions will be critical to uncover the molecular etiology of laminopathies. Here we summarize and critically discuss recent technology to analyze lamina-protein and-chromatin interactions.
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Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently... more
Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.
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The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic... more
The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.
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Research Interests: Molecular Biology, Drosophila melanogaster, Transgenic Mice, Humans, Sequence alignment, and 14 moreMice, Female, Animals, Male, Genes, Muridae, Species Specificity, Amino Acid Sequence, Base Sequence, Fusion Protein, Binding Site, Biochemistry and cell biology, Polycomb group proteins, and Molecular Sequence Data
Philadelphia (Ph)-positive leukemias invariably contain a chromosomal translocation fusing BCR to ABL. The BCR-ABL protein is responsible for leukemogenesis. Here we show that exposure of bcr-null mutant mice to gram-negative endotoxin... more
Philadelphia (Ph)-positive leukemias invariably contain a chromosomal translocation fusing BCR to ABL. The BCR-ABL protein is responsible for leukemogenesis. Here we show that exposure of bcr-null mutant mice to gram-negative endotoxin led to severe septic shock and increased tissue injury by neutrophils. Neutrophils of bcr (-/-) mice showed a pronounced increase in reactive oxygen metabolite production upon activation and were
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A chimeric BCR/ABL oncogene encoding the pl90 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becom ing ill, were found to develop... more
A chimeric BCR/ABL oncogene encoding the pl90 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becom ing ill, were found to develop lymphoblastic leukemia/lymphoma which was transplantable to compatible recipients. Lymphoblasts were ar rested at the pre-B stage of development. Expression of BCR/ABL was not detected in peripheral
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The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog... more
The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, p...
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DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190... more
DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the t...
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Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is... more
Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants. Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts. These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45. These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length. Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin. The DIPs could not be plaque-purified an...
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Chronic myelogenous leukemia is characterized by a specific chromo somal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chlmenc BCR/ABL gene produces a P210 fusion protein with deregulated... more
Chronic myelogenous leukemia is characterized by a specific chromo somal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chlmenc BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains
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Research Interests: Engineering, Physics, Chemistry, Biology, Medicine, and 15 moreMultidisciplinary, Mice, Animals, CpG islands, Early development, Comparative Analysis, Embryonic Stem Cells, Alkaline phosphatase, Histones, Cell Cycle regulation, Large Scale, Gene Expression Analysis, DNA binding proteins, ES cell, and Gene expression profiling
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Research Interests: Genetics, Mice, Female, Animals, Oncogene, and 4 moreMale, Phosphorylation, Clinical Sciences, and Tyrosine
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Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that... more
Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced tri-methylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling.
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The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic... more
The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.
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Our goal was to evaluate intercellular adhesion complex proteins in myocardium in human infarct rupture. Infarct rupture, a fatal complication of myocardial infarction (MI), has been attributed to a defective cell adhesion complex in a... more
Our goal was to evaluate intercellular adhesion complex proteins in myocardium in human infarct rupture. Infarct rupture, a fatal complication of myocardial infarction (MI), has been attributed to a defective cell adhesion complex in a transgenic mouse model. Heart samples were collected from autopsies from infarct rupture and control (nonrupture) MI patients. Both infarcted and remote areas were included. Cell adhesion proteins including alphaE-catenin, beta-catenin, gamma-catenin, and N-cadherin were characterized by immunohistochemistry and immunoblotting. Genetic analysis was undertaken to evaluate mutations and polymorphisms in the alphaE-catenin gene. In addition, infarct rupture was studied in transgenic mice heterozygous for alphaE-catenin C-terminal deficiency, mimicking the situation in human infarct rupture patients. No alphaE-catenin was detected in 70% of remote samples of infarct rupture hearts compared with 20% in control MI by immunohistochemistry. The immunoblot analysis confirmed a significant reduction in remote areas, and complete absence of alphaE-catenin in infarct areas from infarct rupture patients. No mutation or polymorphism of the alphaE-catenin gene was discovered. Other cell adhesion proteins were not significantly affected in remote areas of infarct rupture hearts. Three-fourths of the heterozygous alphaE-catenin C-terminal truncated mice died of infarct rupture, compared with one-fourth of the wild-type littermates. The data show a reduced expression and defective localization of alphaE-catenin in the intercalated disc region in patients dying of infarct rupture. The mechanism of lower expression of alphaE-catenin remains to be elucidated.
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Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated... more
Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.
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To study alterations within the p53 pathway in relation to the development of recurrent stage I endometrioid endometrial carcinoma. Paraffin-embedded tumor tissue of both primary and recurrent tumors from 44 patients with and 44 without... more
To study alterations within the p53 pathway in relation to the development of recurrent stage I endometrioid endometrial carcinoma. Paraffin-embedded tumor tissue of both primary and recurrent tumors from 44 patients with and 44 without recurrence was used for immunohistochemical analysis of TP53, hMdm2, P21(Waf1/Cip1) and M30. DNA was extracted, and mutation analysis of p53 (exon 5-8, 11) was performed by direct sequencing. TP53 overexpression was significantly associated with recurrent disease: Odds Ratio 3.8 (95% CI: 1.5-9.8). Overexpression of TP53 was associated with lower staining indices (SI:0-9) of both hMdm2 and P21 in tumors of patients with recurrence, compared to controls: 2.0 +/- 0.4 vs. 4.0 +/- 0.8 and 1.9 +/- 0.8 vs. 3.6 +/- 0.8, respectively. Eight p53 missense mutations were identified in six patients with recurrence and two controls. One nonsense mutation was found in a patient with recurrence and one deletion in a control patient. Only a minority of TP53 overexpression cases could be explained by the presence of these p53 mutations. TP53 overexpression was significantly predictive for recurrent endometrial carcinoma, and mostly not correlated with p53 mutations. Concomitant low hMdm2 and P21(Waf1/Cip1) expression in tumors with overexpressed TP53 suggests a dysfunctional TP53-P21(Waf1/Cip1) pathway.
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NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood.... more
NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling.
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Research Interests: Biological Sciences, Mutation, Reactive Oxygen Species, Mice, Septic Shock, and 15 moreFemale, Animals, Cell, Male, Actin Cytoskeleton, NADPH oxidase, Neutrophils, Gram-negative bacteria, Lipopolysaccharides, Gene Targeting, Gtp Binding Proteins, Endotoxins, Superoxides, Respiratory Burst, and neutropenia
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Research Interests: Engineering, Gene expression, Protein synthesis, Bone Morphogenetic Proteins, Biological Sciences, and 17 morereal time PCR, Cartilage, Bone, Female, Animals, Post-Transcriptional Gene Regulation, Parathyroid Hormone, Transcription Factor, Chondrogenesis, Osteogenesis, Bone Formation, Rabbits, Bone and Bones, Growth Plate, Vascular Endothelial Growth Factor, Transforming Growth Factor, and Periosteum
Publication View. 34107838. Mouse models in Leukemia / (1995). Voncken, Jan Willem. Abstract. Thesis (doctoral)--Landbouwuniversiteit te Wageningen, 1995. Publication details. Download, http://worldcat.org/oclc/51002264. ...
Research Interests: Genetics, Cancer treatment, Cell Culture, Transgenic Mice, Signal Transduction, and 15 moreHigh Frequency, Cell line, Transgenic Animals, Animal Model, Bone marrow, Karyotype evolution, Spectrum, Mouse Model, Gene Function, Biological Process, Gene Targeting, Transgenic Mouse Technology, Immunoglobulin, Respiratory Burst, and Acute Lymphoblastic Leukemia
CML3 and Ph-positive acute lymphoblastic leukemia are charac terized by a specific chromosomal translocation, t(9;22). The ABL protooncogene from chromosome 9 is juxtaposed to the BCR gene on chromosome 22 by this translocation. The... more
CML3 and Ph-positive acute lymphoblastic leukemia are charac terized by a specific chromosomal translocation, t(9;22). The ABL protooncogene from chromosome 9 is juxtaposed to the BCR gene on chromosome 22 by this translocation. The chimeric BCR/ABL gene produces ...