Biomedical subjects

Manfred Gossen

Publications and source records attributed to Manfred Gossen.

6 recordsLinked to original sources

PubMed2006

Promoter crosstalk effects on gene expression.

Closely spaced transcription signals formally assigned to different, neighboring genes can functionally interact both in their authentic genomic context as well as in engineered transgene constellations. To describe these promoter crosstalk effects quantitatively and qualitatively, we used various combinations of inducible and constitutive expression signals linked in cis. Our results demonstrate that such interactions can be bidirectional, making it difficult to unambiguously assign a particular promoter element exclusively to a specific gene. We show that especially for inducible promoters, crosstalk effects can cause a substantial distortion in the expression of proximal genes, challenging established strategies in generating transgenic animal models and tissue culture systems. Furthermore, these findings provide guidelines for the design of transgenic transcription units that, while refractory to interactions with their chromosomal environment, leave the expression programs of neighboring genes largely untouched.

Animals↗

PubMed2006

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

Animals↗

PubMed2006

A structural role for ATP in the formation and stability of the human origin recognition complex.

The locally restricted recruitment of the multisubunit origin recognition complex (ORC) to eukaryotic chromosomes defines the position of origins of DNA replication. In budding yeast and metazoans the DNA binding activity of ORC is stimulated by ATP and requires an AAA+-type nucleotide binding domain in the largest subunit. Little else is known about the mechanisms behind the ATP requirement for ORC in its initiator function and, specifically, the relevance of nucleotide binding domains present on other subunits. Here we show that ATP is required for specific subunit interactions in the human ORC, with the Orc4 subunit playing a critical role in this dynamic process. ATP is essential for the maintenance of ORC integrity and facilitates complex formation. Thus, besides its previously identified role in DNA binding, ATP serves also as a structural cofactor for human ORC.

Adenosine Triphosphate↗

PubMed2005

Stability and homogeneity of transgene expression in isogenic cells.

Genetically engineered cells are an important tool not only for basic research applications, but also for biotechnology and molecular medicine. Among the issues yet to be solved for this technology are the consequences of randomly integrating DNA into a cell's genome. The problems encountered range from unpredictable expression levels to safety concerns. Recombinase-mediated chromosome engineering is a popular tool for generating stably transfected isogenic cell lines. Using this approach, single-copy integration of foreign DNA fragments can be achieved at predetermined chromosomal loci in the genome. We used such a technology based on the Flp/Flp recombinase target (FRT) recombination system in human 293 cells for comparative promoter studies. The expected integration patterns were obtained with high frequency. In contrast, the phenotypic characterization of expression of the integrated reporter transgene showed remarkable differences between isogenic cell lines, ranging from homogenous expression to mosaic to even complete expression silencing. As long as cell clones with homogenous expression were kept under selective conditions, their expression characteristics could be maintained over a long period. However, this desirable phenotype was progressively lost upon withdrawal of selective pressure. These results were also reflected by the expression instability of the reporter cassette originally inserted in the recipient cell line. Thus, selection for appropriate integration events that ensure reproducible and long-term gene expression has to go beyond the verification of isogenicity.

Cell Line↗

PubMed2003

Functional haplotypes of the RET proto-oncogene promoter are associated with Hirschsprung disease (HSCR).

The activation of the RET signaling pathway during embryogenesis is a crucial prerequisite for a directional migration of enteric nervous system progenitor cells. Loss-of-function germline mutations of the RET proto-oncogene are reported in familial and sporadic cases of Hirschsprung disease (HSCR) with a variable frequency. Furthermore, variants of several RET polymorphisms are over- or under-represented in HSCR populations. Specifically, the c.135A RET variant has been previously shown to be strongly associated with the HSCR phenotype. We have reported an HSCR-phenotype modifying effect of the RET c.135G>A polymorphism due to a within-gene interaction in patients harboring RET germline mutations, yet the function of the c.135G>A variant is unknown. The basic RET promoter region was investigated by DNA sequencing approach in 80 HSCR patients. Identified polymorphisms were genotyped in the HSCR and in a control population and haplotypes were reconstructed. The dual-luciferase assay was used to evaluate the activity of different RET promoter haplotypes. We demonstrate that variants of two RET promoter polymorphisms -5G>A and -1C>A from the transcription start site are associated with HSCR. Furthermore, the -5G>A polymorphism is in strong linkage disequilibrium with the c.135G>A polymorphism. The promoter haplotype -5/-1AC associated with HSCR has a significantly lower activity in an in vitro dual-luciferase expression assay compared with those haplotypes identified in the majority of normal controls. These data suggest a role for RET haplotypes containing the -5A promoter variant in the etiology of HSCR.

Female↗

PubMed2002

Studying gene function in eukaryotes by conditional gene inactivation.

The prospect of specifically controlling gene activities in vivo has become a defining hallmark of many model organisms of biological research. Where once the aim was to gain control over gene activities using endogenous control elements, new technologies have emerged that owe their remarkable specificity to heterologous components derived from evolutionarily distant species. This review highlights inducible transcriptional systems and site-specific recombination. Their quantitative and qualitative characteristics are discussed, with examples of how recent developments have expanded the spectrum of cells and organisms that are now accessible to genetic dissection of unprecedented precision. Transgenesis has already converted the mouse into a prime model for mammalian genetics. Combined with the new approaches of conditional activation or inactivation of genes, this model has opened up new horizons for the analysis of gene function in mammals.

Animals↗