Papers by Dirk Schindelhauer
Human Gene Therapy, Sep 1, 2010
Oncogene, 2011
Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), whic... more Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.
Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer:... more Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the Kurome et al. BMC Biotechnology 2013, 13:43
artificial chromosomes: prospects for defining an optimal centromere
We investigated in different human cell types nuclear positioning and transcriptional regulation ... more We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accor-
Assuming that patterns of sequence variants within highly homogeneous centromeric tandem repeat a... more Assuming that patterns of sequence variants within highly homogeneous centromeric tandem repeat arrays can tell us which molecular turnover mechanisms are presently at work, we analyzed the �-satellite tandem repeat array DXZ1 of one human X chromosome. Here we present accurate snapshots from this dark matter of the genome. We demonstrate stable and representative cloning of the array in a P1 artificial chromosome (PAC) library, use samples of higher-order repeats subcloned from five unmapped PACs (120–160 kb) to identify common variants, and show that such variants are presently in a fixed transition state. To characterize patterns of variant spread throughout homogeneous array segments, we use a novel partial restriction and pulsed-field gel electrophoresis mapping approach. We find an older large-scale (35–50 kb) duplication event supporting the evolutionarily important unequal crossing-over hypothesis, but generally find independent variant occurrence and a paucity of potential ...
The invention relates to a method for producing a DNA construct, whereby two or several DNAs are ... more The invention relates to a method for producing a DNA construct, whereby two or several DNAs are recombined in melted agarose. The invention also relates to vectors which can be used therefor, in addition to a method for providing large DNAs, specially BACs or PACs.
Kunststoffbeutelsystem bestehend aus einem Prozessbeutel und Beutelerweiterungen mit Zugangen (Po... more Kunststoffbeutelsystem bestehend aus einem Prozessbeutel und Beutelerweiterungen mit Zugangen (Ports) fur die Durchfuhrung von Isolierungsverfahren fur Biomolekule im geschlossenen System, Einbringen einer Tragermatrix, Halterahmen, Zulaufbeutel mit Pufferkaskaden fur chemische, biochemische, elektrochemische, physikalische Reinigungsverfahren, einen Elektroelutionsbeutel, Durchfuhrung pulsfeldelektrophoretischer Reinigungen, Elektrodenmaterialien fur die Verwendung innerhalb eines Prozessbeutels, verschiedene Ausfuhrungen und Beispiele fur Genlagerbeutel. Probenentnahme und Verpackung des Produkts im geschlossenen System aus Beutelkomponenten heraus mittels Durchschweistechnik. Durchschweisgerate fur Prozessbeutel, Pulsfeldspannungsgerat und Schaltungen fur einen Reinraumprozessbeutel. Durchschweistechnik fur die Herstellung von Pufferkaskaden in Multikammerbeuteln, die als gemeinsamer Schlauch uber einen Port befullt und sterilisiert werden und anschliesend mittels Peelnahten aufg...
American Journal of Human Genetics, 1994
Patients with X-linked recessive Wiskott-Aldrich syndrome (WAS) manifest eczema, thrombocytopenia... more Patients with X-linked recessive Wiskott-Aldrich syndrome (WAS) manifest eczema, thrombocytopenia and severe immunodeficiency. Mapping studies place the WAS gene locus between the markers TIMP and DXS255 which both have been shown to be recombinant with the disease locus. Linkage analysis in eight families including a large Swiss family showed tight linkage of the disease to the loci DXS255 and DXS1126
Biofutur, 2000
ABSTRACT La manipulation in vivo ou la reconstruction in vitro des chromosomes permettent de mieu... more ABSTRACT La manipulation in vivo ou la reconstruction in vitro des chromosomes permettent de mieux définir leurs éléments fonctionnels ; elles ont créé ainsi des minichromosomes artificiels qui seront peut-être demain les outils privilégiés de la transgénèse.
European journal of medical research, Jan 23, 1998
Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibr... more Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibrosis the hereditary defect is attributed to mutations in one single gene, the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Conventional therapies of CF have dramatically increased the life expectancy of afflicted individuals. However, the ultimate incurability of this disease calls for novel and better therapeutic strategies. As cystic fibrosis is believed to be caused by mutations in one single gene, it has appeared to be the ideal candidate for one of the most tempting approaches in clinical therapy, namely gene therapy. Laboratory protocols for the introduction of genes into various tissues have been developed and applied over the last 15 years. The ease of gene transfer under laboratory conditions gave rise to the hope that rapid advances in gene transfer protocols under clinical settings could be achieved as well. 20 clinical trials of gene therapy...
American journal of human genetics, 1992
A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13... more A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13.2, while at the same time the gene encoding the skeletal muscle ryanodine receptor (RYR1) also has been mapped to this region and has been found to be tightly linked to MHS. RYR1 was consequently postulated as the candidate for the molecular defect causing MHS, and a point mutation in the gene has now been identified and is thought to be the cause of MH in at least some MHS patients. Here we report the results of a linkage study done with 19q12-13.2 markers, including the RYR1 cDNA, in two Bavarian families with MHS. In one of the families, three unambiguous recombination events between MHS and the RYR1 locus were found. In the second family only one informative meiosis was seen with RYR1. However, segregation analysis with markers for D19S75, D19S28, D19S47, CYP2A, BCL3, and APOC2 shows that the crossovers in the first family involve the entire haplotype defined by these markers flanki...
Nucleic Acids Research, 1997
In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centrom... more In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing α satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.
Background Lung treatment with the correct CFTR gene promises a great benefit for the patients. C... more Background Lung treatment with the correct CFTR gene promises a great benefit for the patients. CF is a systemic disease and regulated expression in many cell types is expected to further improve physiology. A regulated gene, unlike a viral construct, does not need tissue specific delivery, because cells normally not expressing CFTR would also not express the therapeutic gene. Moreover, the copy of the gene maintained in a functional form in dividing cells can participate in the natural regeneration of the organ, which may take a considerable period of time after treatment. Thus, the principal aim for a causative therapy is a regulated and stable expression of the normal CFTR gene in many cell types. One major hurdle is the production of a DNA molecule containing all relevant functional elements. This problem is largely independent of the type of delivery, although exceeding the capacity of current viral vectors. Once delivered to the nucleus, the DNA elements will attract cellular ...
Journal of Cystic Fibrosis, 2011
Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of t... more Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra-and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre-and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.
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Papers by Dirk Schindelhauer