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Biomedical subjects

J W Gordon

Publications and source records attributed to J W Gordon.

At least 73 records · Page 4Linked to original sources
PubMed1991

Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice overexpressing a C-terminal fragment of human amyloid precursor protein.

Alzheimer's disease (AD) affects more than 30% of people over 80 years of age. The aetiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated histopathologically with amyloid plaques, neurofibrillary tangles and neuronal loss primarily in the temporal lobe and neocortex of the brain. The core of the extracellular plaque is a derivative of the amyloid precursor protein (APP), referred to as beta/A4, and contains the amino-acid residues 29-42 that are normally embedded in the membrane-spanning region of the precursor. The cellular source of APP and the relationship of its deposition to the neuropathology of AD is unknown. To investigate the relationship between APP overexpression and amyloidogenesis, we have developed a vector to drive expression specifically in neurons of a C-terminal fragment of APP that contains the beta/A4 region, and have used a transgenic mouse system to insert and express this construct. We report here that overexpression of this APP transgene in neurons is sufficient to produce extracellular dense-core amyloid plaques, neurofibrillary tangles and neuronal degeneration similar to that in the AD brain.

Alzheimer Disease↗
PubMed1991

Drug metabolising N-acetyltransferase activity in human cell lines.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by N-acetyltransferase (NAT), a cytosolic enzymic activity which has a wide tissue distribution. Humans can be classified as either fast or slow acetylators on the basis of their ability to metabolise isoniazid or sulphamethazine. These are termed polymorphic substrates. The acetylation of other compounds does not vary amongst individuals, e.g., p-aminobenzoic acid, and are termed monomorphic substrates. NAT from human hepatic and non-hepatic tissues, viz., (i) liver, (ii) the hepatoma cell line HepG2, (iii) tonsil lymphocytes and (iv) the monocytic cell line U937 have been compared with respect to substrate specificity towards polymorphic and monomorphic substrates. The chromatographic and centrifugation behaviour of NAT from these sources has also been investigated. NAT from liver shows 2-fold greater activity towards sulphamethazine than towards p-aminobenzoic acid as substrate. All other cell types tested show at least 70-fold greater activity with p-aminobenzoic as substrate compared to sulphamethazine. NAT from HepG2 cells, U937 cells and tonsil lymphocytes migrates as a single peak during ion-exchange chromatography, whereas the liver NAT activity is separated into two peaks. NAT in HepG2 cells resembles extra-hepatic tissue NAT rather than NAT in liver. HepG2 cells do not therefore represent a good in vitro model for investigation of human metabolism of arylamines or hydrazines. The molecular weight of NAT from U937 cells has been determined by a combination of sucrose density gradient centrifugation and gel filtration to be 31,600 +/- 1200 daltons.

Arylamine N-Acetyltransferase↗
PubMed1991

Isolation and analysis of the mouse genomic sequence encoding Cu(2+)-Zn2+ superoxide dismutase.

Cu(2+)-Zn2+ superoxide dismutase (SOD-1) is a ubiquitously synthesized eukaryotic enzyme with a variety of important roles in mammalian development and physiology. With the goal of better understanding the structure and regulation of this gene, we have cloned and analyzed the genomic structure of the mouse SOD-1 gene. The murine sequence has an intron:exon organization very similar to that of the human gene, though significant diversion was observed in the promoter and 3' regions. These differences account for the observation that only a single transcript for SOD-1 is seen in mouse cells, while two transcripts are derived from the human gene. The cloning of mouse SOD-1 should allow elucidation of the regulatory characteristics of this element, as well as new and informative genetic engineering experiments.

Amino Acid Sequence↗
PubMed1991

Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage.

Immature thymocytes, which coexpress CD4 and CD8, give rise to mature CD4+CD8- and CD4-CD8+ T cells. Only those T cells that recognize self-MHC are selected to mature, a process known as positive selection. The specificity of the T cell antigen receptor (TCR) for class I or class II MHC influences the commitment to a CD4 or CD8 lineage. This may occur by a directed mechanism or by stochastic commitment followed by a selection step that allows only CD8+, class I-specific and CD4+, class II-specific cells to survive. We have generated a mouse line expressing a CD8 transgene under the control of the T cell-specific CD2 regulatory sequences. Although constitutive CD8 expression does not affect thymic selection of CD4+ cells, selection of a class I-specific TCR in the CD8 subset is substantially improved. This outcome is consistent with a model for positive selection in which selection occurs at a developmental stage in which both CD4 and CD8 are expressed, and positive selection by class I MHC generates an instructive signal that directs differentiation to a CD8 lineage.

Animals↗
PubMed1991

Transgenic animals: a new era in developmental biology and medicine.

In the relatively short period since its development, the technique for the production of transgenic animals by pronuclear microinjection, as established by Gordon et al. (1980), has resulted in studies that have clearly had a profound effect on our basic understanding of mammalian development and genetics, and upon our outlook for genetic engineering. The question now is: What remains to be done? Clearly, a greater understanding of the mechanism of integration could lead to more efficient transfer of genes and lessen the problem of rearrangement of host DNA. A better characterization of enhancers and their mode(s) of action would allow design of molecules with highly specific patterns of gene expression, as well as novel distributions of expression. When these goals are met, it may well become scientifically feasible to transfer genes into the human germline. Whether such an undertaking is advisable or even desirable is an issue requiring open discussion, but it is an issue that is clearly distinct from the question of technical feasibility. The most rational approach to this profound question lies in the continued pursuit of knowledge relating to transgenic technology, rather than self-imposed ignorance. Even if never used as a medical therapy, transfer of genes into the germline will reveal what is certainly one of nature's greatest secrets: the nature of the interaction of genes in the development of a multicellular eukaryotic organism. Through the contributions to this volume and in the years ahead, we will find ourselves in a position to unravel the details of the awesome and fascinating phenomenon that is mammalian development.

Animals↗
PubMed1990

Micromanipulation of embryos and germ cells: an approach to gene therapy?

Recent advances in mammalian gamete and embryo micromanipulation have stimulated the scientific and medical communities, and to some degree the public at large, to become aware that treatment of genetic disease by direct alteration of the genetic code may soon be possible. Because these micromanipulation techniques result in modification of the genotype at the earliest stages of development, such "gene therapy" affects not only the conceptus itself but also its germ cells. Thus such genetic modifications are heritable and can be transmitted indefinitely to succeeding generations of progeny. In the presentation, both narrow and broad definitions of gene therapy will be considered with respect to the techniques upon which they are based, their potential for treatment of genetic disease, and their current feasibility.

Animals↗
PubMed1990

Analysis of the hotfoot (ho) locus by creation of an insertional mutation in a transgenic mouse.

Hotfoot (ho) mutation is a recessive trait in mice, characterized by motor disorder and male sterility, that maps to chromosome 6. We have identified a transgenic mouse pedigree with a similar trait. Using genetic and molecular approaches, we have demonstrated that the foreign DNA element is located in or near the ho locus. This new allele, designated hoJwg and presumably created by insertional mutagenesis, should make it possible to clone the ho gene. Male infertility in hoJwg male homozygotes was determined to be due to inability of sperm to penetrate the zona pellucida. This was demonstrated by rescuing mutant males by a new technique of gamete micromanipulation, zona pellucida drilling. These findings show that zona drilling is useful both for analysis and preservation of animals with reduced male fertility.

Animals↗
PubMed1990

Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos.

The polymerase chain reaction (PCR) technique has been adapted to identify the sex of preimplantation mouse embryos rapidly. PCR was used to amplify a specific repeated DNA sequence on the Y chromosome from a single isolated blastomere in under 12 hr. The remainder of the biopsied embryo was then transferred to a pseudopregnant female and carried to term. Using this technique, 72% of embryos can be classed as potentially either male or female. Transfers of such embryos have produced pregnancies with 8/8 fetuses (100%) being of the predicted sex. Variations of the technique have demonstrated certain limitations to the present procedure as well as indicated possible strategies for improvement of the assay. The PCR technique may have wide application in the genetic analysis of preimplantation embryos.

Animals↗
PubMed1990

Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene.

Transgenic mice carrying the activated N-ras oncogene under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. The transgene is expressed in a tissue distribution consistent with the fact that it is driven by the MMTV-LTR, and similarly to MMTV/H-ras constructs, its presence elicits tumors in Harderian, mammary and salivary glands. In addition it appears to compromise male reproductive function, which has not been described with the other ras transgenes. This finding is consistent with the existence of distinct physiological actions for each of the ras family members.

Animals↗
PubMed1990

Clinical evaluation of three approaches to micromanipulation-assisted fertilization.

Three different micromanipulation procedures were used to assist human fertilization in cases of severe male factor infertility. Zona drilling was performed either with acid Tyrode's solution, mechanically following zona softening with chymotrypsin, or by partial zona dissection. The fertilization rate was lowest in the zona drilling/acid Tyrode's group (7/40; 17.5%), although no differences between groups (zona drilling/chymotrypsin: 21/84, 25%; partial zona dissection: 31/143, 21.7%) were significant. The fertilization rate was significantly increased relative to untreated eggs from the same patients only in the partial zona dissection group (31/143, 21.7% versus 4/102, 3.9%). Oocyte damage occurred at a high rate as a result of zona drilling with acid Tyrode's solution (13/41, 37%). Embryonic development was compromised after zona drilling with chymotrypsin: only 7/12 (58.3%) of the fertilized oocytes cleaved, and the morphology of many of the cleaved embryos was abnormal. Although only 61% (16/26) of the diploid embryos resulting from partial zona dissection cleaved, the embryonic morphology of these embryos was comparable with controls. No pregnancies resulted from the transfer of manipulated embryos. We conclude that although zona manipulation increases the fertilization rate, losses due to oocyte trauma, low rates of diploid fertilization, low rates of cleavage, and a high frequency of abnormal cleavage reduce the number of embryos available for transfer.

Dissection↗
PubMed1989

Expression of a methotrexate resistant dihydrofolate reductase gene in transgenic mice.

We have previously demonstrated systemic resistance to methotrexate (MTX) in transgenic mice carrying a foreign, mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3) gene. The new gene was introduced as a cDNA cloned into an expression vector driven by the simian virus 40 (SV40) early promoter. Previous physiologic studies suggested that transgenic mice tolerated drug doses invariably lethal to controls on the basis of gastrointestinal (GI) resistance to MTX. In the present study we evaluated foreign gene expression at the RNA level in the three major sites of MTX toxicity: intestine, liver, and bone marrow. The transgene was transcriptionally active in small bowel, and levels of expression were high in animals tolerating the largest doses of MTX. The gene was also expressed in the liver in some pedigrees, but was not detected in hemopoietic tissues of any of the pedigrees tested. Our studies correlate the site of expression of a drug resistant dhfr gene with an altered physiologic response to MTX, and demonstrate that transgenic mice can be used as a test system for expression of genes considered for use in somatic gene therapy.

Animals↗
PubMed1989

Association of foreign DNA sequence with male sterility and translocation in a line of transgenic mice.

We have analyzed a line of transgenic mice derived from injection of a cloned human interferon cDNA. This line manifests total male sterility of males carrying the human sequence, while male littermates not harboring the foreign DNA are fertile. All females are fertile. Karyotypes of transgenic animals show 2:12 translocation. The microinjected sequence maps to one of the translocation chromosomes composed of a large portion of chromosome 12 to which has been translocated a segment of chromosome 2. Analysis of the sterile males reveals significant abnormalities of spermatogenesis and faulty chromosome synapsis that involves the translocation chromosomes. These findings show that transfer of foreign DNA into mouse embryos may lead to chromatin breakage and infertility of transgenic animals.

Animals↗
PubMed1989

Successful microsurgical removal of a pronucleus from tripronuclear human zygotes.

We have microsurgically extracted a pronucleus from 11 tripronuclear human zygotes under two different experimental conditions. One group of six zygotes was incubated in cytochalasin D for 30 minutes before manipulation. The remaining five zygotes were manipulated without preincubation in cytoskeletal relaxing agents. When cytoskeletal relaxants were used, all six embryos survived manipulation and cleaved. One embryo arrested spontaneously at the two-cell stage; all others cleaved to at least four cells, and one embryo reached the advanced morula stage. Of the five eggs manipulated without prior cytochalasin D and colcemid treatment, none survived for more than 30 minutes after manipulation. In none of the tripronuclear eggs was it possible to definitely identify the male pronuclei. Because development was interrupted for karyotyping, it is likely that these embryos would have otherwise continued to develop to more advanced stages. These results indicate that it is possible to microsurgically "epronucleate" tripronuclear human zygotes and obtain further development and that use of cytoskeletal relaxants facilities this manipulative procedure. However, because epronucleated embryos have significant developmental potential, chromosome studies are needed to confirm complete removal of a male pronucleus before such techniques can be applied clinically.

Cytochalasin D↗
PubMed1989

Insertional mutation in a transgenic mouse allelic with Purkinje cell degeneration.

Purkinje cell degeneration (pcd) is an autosomal recessive mutation which maps to chromosome 13 in the mouse. The pcd mutation causes loss of cerebellar Purkinje cells, retinal photoreceptor cells, and olfactory bulb mitral cells, as well as abnormalities of spermatogenesis. pcd is among a number of autosomal recessive mutations in mice and humans that affect neurologic function and male fertility. The cloning of one or more of these loci would contribute significantly to our understanding of the genetic control of development and maintenance of affected cell types. We report here identification of a transgenic mouse line with an insertional mutation that is allelic with pcd and manifests histopathologic features indistinguishable from those of the spontaneous mutation. The creation of an allele of pcd by transgene insertion should make possible the cloning of the pcd locus.

Alleles↗