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S O'Gorman

Publications and source records attributed to S O'Gorman.

17 recordsLinked to original sources
PubMed1998

Selection of primary cell cultures with cre recombinase induced somatic mutations from transgenic mice.

Deletion of genes in defined cell types has been achieved using a combination of gene targeting techniques and the Cre- lox P recombination system. Here we present a method to selectively isolate genetically altered primary cell cultures based on the permanent activation of a drug-resistance gene by the Cre recombinase. Transgenic mice were generated harboring a dormant form of the hygromycin resistance gene. This mouse line was crossed with mice carrying a constitutive Cre gene and an endogenous floxed allele. Primary fibroblasts established from triple transgenic embryos displayed not only hygromycin resistance but also recombination of the endogenous floxed allele. These results prove the potential of this approach.

Animals↗
PubMed1997

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells.

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.

Alleles↗
PubMed1997

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.

Animals↗
PubMed1991

Recombinase-mediated gene activation and site-specific integration in mammalian cells.

A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter. By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site. The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations.

Animals↗
PubMed1991

A safe new procedure for high-risk patients with symptomatic gallstones.

Cholecystectomy is associated with an appreciable mortality rate in elderly high-risk patients. Patients aged over 60 years with symptomatic gallstones, at high operative risk, underwent cholecystotomy under local anaesthesia through a 3-cm incision. Stones were removed and clearance was demonstrated endoscopically and by tube cholecystography. Catheter drainage was continued for 7 days until a further cholecystogram confirmed clearance. The procedure was attempted in 26 patients with concomitant cardiovascular, respiratory or malignant disease. Successful removal of all gallbladder stones was possible in 24 patients. Four patients had common bile duct stones demonstrated on cholecystography, all of which were successfully treated by endoscopic sphincterotomy. All patients are symptom-free at a mean follow-up of 36 weeks with no recurrent stones on ultrasonography.

Aged↗
PubMed1991

Cystic duct obliteration and gallbladder mucosal destruction: a feasible alternative to cholecystectomy.

'Chemical cholecystectomy' has been proposed as an alternative to removal of the gallbladder. This study assessed cystic duct obliteration using bipolar electrocoagulation (with sham cannulation controls) and gallbladder mucosal treatment with tetracycline (or saline controls) in 29 mongrel dogs. Cystic duct obstruction was assessed by tube cholecystography at day 14, and epithelial damage by histology at day 42. Electrocoagulation by duct diathermy effectively occluded the cystic duct in 14 of 19 animals; however, this was associated with mucocele formation unless mucosal treatment with tetracycline was also performed. Immediate instillation of tetracycline after duct electrocoagulation produced only partial epithelial damage. The combination of duct electrocoagulation and delayed tetracycline instillation at 14 days produced complete destruction of all gallbladder epithelium and an effective chemical cholecystectomy.

Animals↗
PubMed1991

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

Animals↗
PubMed1990

Three-dimensional autoradiographic localization of quench-corrected glycine receptor specific activity in the mouse brain using 3H-strychnine as the ligand.

The autoradiographic analysis of neurotransmitter receptor distribution is a powerful technique that provides extensive information on the localization of neurotransmitter systems. Computer methodologies are described for the analysis of autoradiographic material which include quench correction, 3-dimensional display, and quantification based on anatomical boundaries determined from the tissue sections. These methodologies are applied to the problem of the distribution of glycine receptors measured by 3H-strychnine binding in the mouse CNS. The most distinctive feature of this distribution is its marked caudorostral gradient. The highest densities of binding sites within this gradient were seen in somatic motor and sensory areas; high densities of binding were seen in branchial efferent and special sensory areas. Moderate levels were seen in nuclei related to visceral function. Densities within the reticular formation paralleled the overall gradient with high to moderate levels of binding. The colliculi had low and the diencephalon had very low levels of binding. No binding was seen in the cerebellum or the telencephalon with the exception of the amygdala, which had very low levels of specific binding. This distribution of glycine receptors correlates well with the known functional distribution of glycine synaptic function. These data are illustrated in 3 dimensions and discussed in terms of the significance of the analysis techniques on this type of data as well as the functional significance of the distribution of glycine receptors.

Animals↗
PubMed1988

Histopathologic findings in Best's vitelliform macular dystrophy.

Postmortem donor eyes from a 69-year-old man with Best's vitelliform macular dystrophy showed retinal pigment epithelial cells across the entire fundus that had accumulated an excessive amount of lipofuscin as defined by ultrastructural appearance, autofluorescence studies, and staining properties. Lipofuscin accumulation was particularly notable in some pigment epithelial cells in the fovea. An accumulation of heterogeneous material located between Bruch's membrane and the pigment epithelium in the fovea was believed to represent the location of a previtelliform lesion. This material appeared to be derived from degenerating pigment epithelial cells and contained few intact lipofuscin granules. Foveal photoreceptor loss occurred above the lesion and in midperipheral sites where the subretinal space contained collections of outer segment debris and phagocytic cells. Best's vitelliform macular dystrophy appears to be a generalized disorder of the pigment epithelium that secondarily affects focal areas of the retina.

Adult↗
PubMed1987

Healing and growth of half-eye "compound eyes" in Xenopus: application of an interspecific cell marker.

Surgically constructed compound eyes have been widely used to examine the development of retinotectal projections in amphibians. Such studies have been limited, however, by the lack of an adequate cellular marker with which to assess the contributions of grafted and host tissues to the later larval and adult retina. We have followed the growth of graft- and host-derived ocular tissues in interspecific compound eyes prepared by orthotopic and heterotopic exchanges of half-eye fragments between pigmented Xenopus borealis and albino Xenopus laevis embryos. This genotypic combination allowed the growth of graft-derived choroid and pigment epithelium to be studied in the living animal, and permitted cell-by-cell resolution of graft- and host-derived neurons in quinacrine-stained paraffin sections. At mid- and late-larval stages graft-derived neurons occupied large and usually coherent territories of retina in all classes of orthotopic and heterotopic compound eyes. In successfully healed cases, sample means of the percentage of the total retina occupied by graft-derived neurons ranged from 43 to 51%. Graft-derived territories originated near the optic nerve head and extended into the germinal neuroepithelium at the retinal periphery. As compared to orthotopic compound eyes, graft-derived territories in all classes of heterotopic compound eyes were slightly smaller and significantly more variable. Despite this variability, the correlation between graft-derived pigment epithelial and retinal territories was quite good in heterotopic compound eyes. While graft-host borders were generally sharp, there was also evidence of local cell mixing over distances of several cell diameters in the inner nuclear and ganglion cell layers. Single displaced cells, isolated from other members of their genetic cohort by 5 or more cell diameters, were also present in the inner nuclear and ganglion cell layers.

Animals↗
PubMed1985

Degeneration of thalamic neurons in "Purkinje cell degeneration" mutant mice. I. Distribution of neuron loss.

The Purkinje cell degeneration (pcd) mutation of the mouse is an autosomal recessive allele which previous studies have shown to be the cause of rapid degeneration of nearly all cerebellar Purkinje cells between 18 and 30 postnatal days of age (P18-P30), and slowly developing, progressive losses of retinal photoreceptor cells and mitral cells of the olfactory bulb. Through examination of serial frozen sections alternately stained for Nissl substance and for degenerating neuronal processes, we have found that discrete populations of thalamic neurons degenerate rapidly between P50 and P60. Severely affected nuclei, in which a majority of neurons degenerate, include the central division of the mediodorsal nucleus, the ventral medial geniculate, posterior, posterior ventromedial, and submedial nuclei, and those portions of the ventrolateral and posteromedial nuclei which immediately surround the medial division of the ventrobasal complex. More subtle cell losses occur during the same time period in restricted portions of the lateral ventrobasal, dorsal lateral geniculate, and lateral posterior nuclei, but even at P180 these nuclei are not markedly atrophic. No common denominator among target cell populations has been established. The pcd allele affects a diverse assortment of specific relay nuclei; degeneration has not been recognized in thalamic nuclei characterized primarily or exclusively by subcortical projections or by cortical projections directed relatively selectively to superficial or deep cortical laminae. The neuronal degenerations in the thalamus are not precipitated by prior or concurrent degeneration of cortical targets or afferent sources, though striking transneuronal changes, including cell death, do develop following thalamic neuronal degeneration in this mutant. No previously described murine mutant phenotype includes the rapid degeneration of highly restricted neuronal populations beginning at these relatively advanced ages.

Aging↗
PubMed1985

Degeneration of thalamic neurons in "Purkinje cell degeneration" mutant mice. II. Cytology of neuron loss.

The cytology of thalamocortical relay neuron degeneration in the ventral medial geniculate nucleus (vMG) of mice homozygous for the autosomal recessive Purkinje cell degeneration (pcd) mutation has been studied by light and electron microscopy. More limited sampling of the submedial and mediodorsal nuclei suggested that cytological alterations in the vMG were typical of all degenerating thalamic nuclei. The number of vMG neurons in pcd mutants was comparable to controls at and prior to postnatal day 40 (P40). By P60 seventy percent, and by P90 approximately 90%, of the original complement of vMG neurons had degenerated in mutant mice. At P30, the general cytological organization of vMG neurons closely resembled that of neurons in littermate (+/+ or +/pcd) controls, but neurons in mutants were distinguished by the presence of small aggregates of fine granules (approximately 9 nm in diameter) that were commonly associated with otherwise normal cisternae of rough endoplasmic reticulum; neither the number nor the size of these granular aggregates increased in older mutants. By P50 cytoplasmic organelles were curiously distributed in more severely affected neurons: large areas of cytoplasm were occupied exclusively by polysomes, while profiles of endoplasmic reticulum and the Golgi apparatus appeared to be reduced. Before frank degenerative changes were apparent (at P50), all classes of synaptic terminals identified in normal mice were found to have made morphologically normal synaptic contacts on mutant vMG neuron dendrites. In contrast to the homologous nuclear complex in the cat, presynaptic dendrites were not apparent in synaptic glomeruli in wild-type or mutant murine vMG. Cytopathological alterations in the neuropil of P50 and older mutants were dominated by degenerating dendritic profiles; there was no evidence that the loss of thalamic neurons in pcd mutants was associated with synaptic agenesis or dysgenesis or the prior or concurrent degeneration of afferent synaptic terminals.

Aging↗
PubMed1985

Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitis pigmentosa.

The composition and amount of vitamin A stored in the retinal pigment epithelium and choroid (RPE-Ch) was evaluated in postmortem donor eyes from a patient with retinitis pigmentosa that was probably inherited by an autosomal recessive mode. Additionally, the soluble proteins in the neural retina and RPE-Ch cytosols and interphotoreceptor matrix were examined collectively for the presence of interstitial retinol-binding protein (IRBP). Although there was depletion of the amount of vitamin A stored in the RPE, this was commensurate with the histopathologic findings on the RPE extent and thickness. No evidence was found for an accumulation of free retinol. Nearly all of the vitamin A stored in the RPE was esterified. As in normal eyes, the retinyl esters consisted mainly of palmitate mixed with a small proportion of stearate. Eleven-cis retinyl esters were present, although their proportion was lower than that reported for normals. IRBP could not be detected in stained gels of the soluble proteins, or by autoradiography of these gels after treatment with 125I-concanavalin A. These findings suggest that depletion of stored vitamin A, accumulation of free retinol, or deficiency of 11-cis isomer are unlikely to be causative factors in the retinal degeneration examined here. Although the depletion of IRBP seen at this advanced stage might be secondary to the advanced loss of photoreceptors, the authors cannot rule out the possibility that a relative deficiency or abnormality in this protein at earlier disease stages may contribute to the pathogenesis of retinitis pigmentosa.

Aged↗
PubMed1979

Neurochemical and morphological consequences of axon terminal degeneration in cerebellar deep nuclei of mice with inherited Purkinje cell degeneration.

The concentrations of free amino acids and the activities of transmitter-related enzymes, glutamic acid decarboxylase (GAD), choline acetylase (ChAC) and GABA-transaminase (GABA-t) were measured in cerebellar cortex and deep cerebellar nuclei from the mouse mutant Purkinje cell degeneration (pcd) at various times before and after Purkinje cell loss. Axosomatic synapses on target cells in pcd deep nuclei were quantified by electron microscopy during and after degeneration. The concentration of GABA (nmol/mg wet weight), the Purkinje cell transmitter, was normal in pcd cerebellar cortex and deep nuclei before onset of Purkinje cell degeneration on postnatal day 15. Just after the major period of Purkinje cell loss in cerebellar cortex, GABA concentration was unchanged in the cortical layers but fell to 50% of normal values in the deep nuclei of pcd animals killed either by decapitation or by microwave irradiation. No other measured free amino acid decreased. There were no long-term increases following Purkinje cell degeneration in the concentration of any transmitter amino acids or related enzymes, GAD, ChAC or GABA-t, and thus no indication of axonal sprouting reactions. Progressive losses occurred in wet weight and protein and in activity of GABA-t in both the cerebellar cortex and the deep nuclei of pcd animals. Electron microscopic analysis indicated that Purkinje cell axon terminals contact 30% or more of the somatic surface of principal neurons of the lateral nucleus of the normal cerebellum, but only about 2% of the corresponding sites in the pcd cerebellum. Glial leaflets, rather than other synaptic terminals take their place. Axon terminals may degenerate earlier than Purkinje somata in the pcd disease.

4-Aminobutyrate Transaminase↗
PubMedDate not supplied

Reassessment of cerebral capillary changes in acute global ischemia and their relationship to the "no-reflow phenomenon".

Electron and light microscopic studies were performed on rabbit brain to re-examine the structural changes of endothelial cells and perivascular glia following ischemia. Although swelling of perivascular glia occurred, earlier findings of extreme perivascular glial swelling and bleb formation leading to luminal collapse and plugging could not be confirmed. Ischemic brains, however, had a higher proportion of small-diameter capillaries than controls. It is felt that structural changes in ischemic capillary walls in themselves are not sufficient to explain failed cerebral reperfusion, or the no-reflow phenomenon.

Animals↗