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R Feil

Publications and source records attributed to R Feil.

At least 19 recordsLinked to original sources
PubMed2007

Conditional somatic mutagenesis in the mouse using site-specific recombinases.

In the last decade, site-specific recombinases (SSRs), such as Cre and Flp, have emerged as indispensable tools for the precise in vivo manipulation of the mouse genome. It is now feasible to control, in space and time, the onset of gene knockouts in almost any tissue of the mouse, thus greatly facilitating the creation of sophisticated animal models for human disease and drug development. This review describes the basic principles and current status of the SSR technology, with a focus on strategies for conditional somatic mutagenesis using the Cre/lox system and ligand-activated Cre recombinases. Practical hints for generating and analysing conditional mouse mutants will be given and exciting novel applications of the SSR technology will be discussed, such as cell fate mapping and the combined use of Cre, Flp and other biotechnological tools. It will be shown how genetic manipulation of the mouse by site-specific recombination can provide new solutions to old problems in the analysis of human physiology and pathophysiology and how it can be employed for drug discovery and development.

Animals↗
PubMed2006

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

Animals↗
PubMed2006

Limited evolutionary conservation of imprinting in the human placenta.

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

Alleles↗
PubMed2006

[The clinical use of the ISO-C(3D) imaging system in calcaneus fracture surgery].

We compared in a prospective study including 82 patients treated with ORIF of an intraarticular calcaneus fracture the quality of fluoroscopy, intraoperatively Iso-C(3D) and postoperative CT-scans. Therefore the posterior facet of the calcaneus (PFOC) was divided into three sectors. Joint steps and fracture gaps were detected by two independent investigators and statistically analysed. Another focus was to evaluate if the findings due to intraoperatively Iso-C(3D) assessment performed by the surgeon were correct and subsequently influenced the surgical procedure. There were no statistically differences between the Iso-C(3D)- and CT findings concerning joint steps or fracture gaps in PFOC sectors I-III. With fluoroscopy an assessment of the PFOC sectors I and II was not possible. In six cases (7.3%), intraoperative reduction was redone after performing an Iso-C(3D) scan. In ten cases, 12 malpositioned screws were replaced (12.2%/14.6%). These results suggest that intraoperative 3D Iso-C(3D) imaging provides a high diagnostic reliability. By careful assessment of the images the surgeons receive information which could lead to a change of the operative strategy.

Algorithms↗
PubMed2006

Function of cGMP-dependent protein kinases as revealed by gene deletion.

Over the past few years, a wealth of biochemical and functional data have been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGK proteins generated by molecular biology techniques yielded important biochemical knowledge, such as the function of the NH(2)-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, and the substrate specificity of the enzymes. Genetic approaches have proven especially useful for the analysis of the biological functions of cGKs. Recently, some of the in vivo targets and mechanisms leading to changes in neuronal adaptation, smooth muscle relaxation and growth, intestinal water secretion, bone growth, renin secretion, and other important functions have been identified. These data show that cGKs are signaling molecules involved in many biological functions.

Animals↗
PubMed2006

Genomic imprinting in the placenta.

Genomic imprinting is an epigenetic mechanism that is important for the development and function of the extra-embryonic tissues in the mouse. Remarkably all the autosomal genes which were found to be imprinted in the trophoblast (placenta) only are active on the maternal and repressed on the paternal allele. It was shown for several of these genes that their paternal silencing is not dependent on DNA methylation, at least not in its somatic maintenance. Rather, recent studies in the mouse suggest that placenta-specific imprinting involves repressive histone modifications and non-coding RNAs. This mechanism of autosomal imprinting is similar to imprinted X chromosome inactivation in the placenta. Although the underlying reasons remain to be explored, this suggests that imprinting in the placenta and imprinted X inactivation are evolutionarily related.

Animals↗
PubMed2005

Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIalpha isoform is prominent in the cerebellum and medulla, whereas the cGKIbeta isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Animals↗
PubMed2004

Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.

Animals↗
PubMed2002

Differential patterns of histone methylation and acetylation distinguish active and repressed alleles at X-linked genes.

In female mammals, one of the two X chromosomes is inactivated to compensate for the difference in dosage of X-linked genes between males and females. X inactivation involves sequential alterations to the chromatin that ultimately lead to the transcriptional repression of genes on the X chromosome. Here, histone methylation and acetylation along X-linked genes are investigated by chromatin immunoprecipitation (ChIP) of adult fibroblast cell lines. At PGK1 and HPRT, chromatin on the active X chromosome reveals H3 lysine 4 methylation and acetylation of histones H3 and H4. These modifications are absent on the repressed allele, which is marked by H3 lysine 9 methylation. On the expressed allele of XIST (on the inactive X chromosome), we found that H3 acetylation is confined to the promoter, whereas H3 lysine 4 methylation and H4 acetylation are present along the entire gene. On the repressed XIST allele, in contrast, the promoter and gene exhibit H3 lysine 9 methylation. At only 1.5 kb upstream of the XIST gene, chromatin on the inactive X chromosome has strongly reduced levels of H4 acetylation and is marked by both H3 lysine 9 and H3 lysine 4 methylation. These data demonstrate that patterns of histone methylation and acetylation are distinct along and upstream of XIST and suggest that the inactive X chromatin configuration occurs at a region close to the 5' end of the gene.

Acetylation↗
PubMed2001

Environmental effects on genomic imprinting in mammals.

Genomic imprinting is an epigenetic marking mechanism by which certain genes become repressed on one of the two parental alleles. Imprinting plays important roles in mammalian development, and in humans its deregulation may result in disease and carcinogenesis. During different medical, technological and scientific interventions, pre-implantation embryos and cells are taken from their natural environment and subjected to culture in artificial media. Studies in the mouse demonstrate that environmental stress, such as in vitro culture, can affect the somatic maintenance of epigenetic marks at imprinted loci. These effects are associated with aberrant growth and morphology at fetal and perinatal stages of development.

Animals↗
PubMed2001

Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes.

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene MEST: Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.

Animals↗
PubMed2001

DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1.

The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.

Acetylation↗
PubMed2001

Probing chromatin structure with nuclease sensitivity assays.

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

Animals↗
PubMed2000

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse.

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

Alleles↗
PubMed2000

Temporally controlled somatic mutagenesis in smooth muscle.

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

Animals↗