http://repub.eur.nl/res/aut/6044/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/2487/ 1993-01-01T00:00:00Z We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression. http://repub.eur.nl/res/pub/2430/ 1990-01-01T00:00:00Z Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells. http://repub.eur.nl/res/pub/2448/ 1990-01-01T00:00:00Z We have exploited the structural homology, but different patterns of expression of the murine and human Thy-1 genes to map a number of tissue-specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally-regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent. http://repub.eur.nl/res/pub/2455/ 1990-01-01T00:00:00Z A 21.5 kb DNA fragment carrying the entire chicken lysozyme gene locus was introduced into the germ line of mice. The fragment contains the transcribed region plus 11.5 kb 5'-flanking and 5.5 kb 3'-flanking sequences including all known cis-regulatory elements and the 5' and 3' attachment elements (A-elements) which define the borders of the DNase I sensitive chromatin domain. All sequences which adopt a DNase I hypersensitive chromatin conformation in vivo are present on the construct. Seven founder mice were analysed. All of these expressed chicken lysozyme RNA at high levels specifically in macrophages, as is the case in the donor species. Expression levels are dependent on the copy number of integrated genes indicating that a complete gene locus, as defined by its chromatin structure, functions as an independent regulatory unit when introduced into a heterologous genome. http://repub.eur.nl/res/pub/2439/ 1989-01-01T00:00:00Z The regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell lines and transgenic animals has turned out to be at a low level relative to that of endogenous genes, and influenced by the chromosomal site of insertion of the exogenous construct. We have previously shown that if regions flanking the human beta-globin locus are introduced into the mouse genome along with the human beta-globin gene, a level of expression comparable to that of endogenous genes can be achieved that is also independent of integration site. We have now defined a dominant control region with these properties consisting of 6.5 kilobases of DNA encompassing erythroid cell-specific DNase I hypersensitive sites. The identification of such dominant control regions could have important applications in somatic gene therapy. http://repub.eur.nl/res/pub/2444/ 1989-01-01T00:00:00Z Using the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of the endogenous mouse alpha-globin genes. Transgenic fetuses with high-copy numbers of the transgene suffer severe anemia and die before birth. Using a construct with both the human alpha- and beta-globin genes and the beta-globin DCR, live mice with low-copy numbers were obtained. Both human globin genes are expressed at high levels in adult red cells to give human hemoglobin HbA in amounts equal to or greater than endogenous mouse hemoglobin. Expression of HbA in murine red cells is not accompanied by any increase in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). However, these transgenic mice tend to have an increased number of reticulocytes in peripheral blood; consistent with some degree of hemolysis. Metabolic labeling experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with an alpha/beta biosynthetic ratio of approximately 0.6. Thus, these mice have mild anemia. These results are discussed with relation to the coordinate regulation of alpha- and beta-globin synthesis in erythroid tissues.