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J A Wolff

Publications and source records attributed to J A Wolff.

At least 73 records · Page 4Linked to original sources
PubMed1993

Particle bombardment-mediated gene transfer and expression in rat brain tissues.

We have previously demonstrated that the particle bombardment method for gene transfer (Accell) provides a new means for transfection of various cell types in culture. In this study we evaluate its application to rat brain systems. Using a luciferase (luc) gene as a reporter, we obtained high levels of transient gene expression in primary cultures of fetal brain tissue. Reduced but significant levels were also detected in adult brain primary cultures. Both neuron and glial cells were transfected using this technique. The transient gene expression level obtained with Accell was at least 100-fold higher than that obtained with three other gene transfer methods. The relative strengths of four cellular and seven viral promoters were also evaluated in these cultures. In vivo gene expression was studied using freshly excised and bombarded fetal brain tissues which were immediately transplanted into caudate or intracortical brain tissues of adult host animals. Assays showed that luciferase activity was present in transplants for up to two months following gene transfer. In vitro and in vivo expression of a rat tyrosine hydroxylase (TH) gene, a candidate gene for treatment of Parkinson's disease, was also detected in this rat brain system. Our results suggest that the particle bombardment gene transfer technology can be employed as an effective method for ex vivo gene transfer into brain tissues.

Animals↗
PubMed1993

Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection.

Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in mdx mouse muscle following intramuscular plasmid DNA injection. Intramuscular injection of a single plasmid DNA encoding both luciferase and dystrophin resulted in stable luciferase expression for at least 2 months in mdx muscle, whereas injection of plasmid DNA encoding only luciferase did not result in stable luciferase expression. These results suggest that expression of either full-length or Becker-like dystrophins protects mdx mouse myofibers from degeneration.

Animals↗
PubMed1993

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term.

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

Animals↗
PubMed1993

Gene therapy--a primer.

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Acquired Immunodeficiency Syndrome↗
PubMed1992

sar: a genetic mouse model for human sarcosinemia generated by ethylnitrosourea mutagenesis.

A mouse mutant with sarcosinemia was found by screening the progeny of ethylnitrosourea-mutagenized mice for aminoacidurias. Paper chromatography, column chromatography, and gas chromatography-mass spectrometry identified high levels of sarcosine in the urine of the mutant mice. While sarcosine cannot be detected in the urine of plasma of normal mice, the urinary sarcosine level of 102 +/- 58 mmol per g of creatinine in the mutant mice was at the upper range of the urinary levels (1.5-4.5 mmol of sarcosine per g of creatinine) observed in humans with sarcosinemia. Similarly, the plasma sarcosine level of 785 +/- 153 mumol/liter in the sarcosinemic mice was at the upper range of the plasma sarcosine levels (53-760 mumol/liter) observed in affected humans. Sarcosine dehydrogenase [sarcosine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.1] activity was deficient in sarcosinemic mice. The sarcosinuria phenotype in these mice was inherited as an autosomal recessive trait. This mouse mutant provides a useful genetic model for human sarcosinemia and for development of therapeutic approaches for genetic disease.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Long-term survival of autologous muscle grafts in rat brain.

The ability of muscle to persist in the brain was explored. Pieces of rat quadriceps muscles were removed, minced and transplanted back into the same animal's cortex. Brains implanted with minced muscle consistently contained substantial grafts for at least 6 months. Histological, immunohistochemical, and electron microscopic examination indicated that the muscle grafts after 6 months contained healthy and well-differentiated myofibers. The long-term survival of muscle cells suggested that this technique will be useful for studies of brain and muscle and for the development of new therapeutic modalities.

Animals↗
PubMed1992

Intracerebral transplants of primary muscle cells: a potential 'platform' for transgene expression in the brain.

After the transplantation of rat primary muscle cells into the caudate or cortex of recipient rats, the muscle cells were able to persist for at least 6 months. Muscle cells transfected with expression plasmids prior to transplantation were able to express reporter genes in the brains for at least 2 months. These results suggest that muscle cells might be a useful 'platform' for transgene expression in the brain.

Animals↗
PubMed1992

Persistence of plasmid DNA and expression in rat brain cells in vivo.

The purpose of this study was to determine whether plasmid DNA is able to persist in nondividing or slowly dividing brain cells in vivo. A new cationic lipid formulation which contains 70 mol% of DOTMA (N[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium) and 30 mol% of cholesterol was used to transfect reporter genes into fetal brain cells in culture that were then transplanted into adult host brains. Gene expression was localized both to glial and neuronal cells after transfection of fetal brain cells with pRSVLac-Z, the gene coding for Escherichia coli beta-galactosidase protein. After the transfection of pRSVL plasmid which contains the firefly luciferase gene into fetal brain cells that were transplanted, substantial amounts of luciferase and pRSVL DNA were present in the host brains for 1 to 2 months. These results have implications for intracerebral viral infections and gene therapy of brain disorders.

Animals↗
PubMed1992

Direct gene transfer into nonhuman primate myofibers in vivo.

Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly. This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids. A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys. After the injection of the E. coli beta-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers. The luciferase reporter gene in pRSVL was used to quantify the amount of expression. The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline. Luciferase activity persisted for at least 4 months after injection. However, the luciferase expression was considerably less than that in rodents. Preliminary studies explored why expression was less in monkeys. Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents. This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers. Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys.

Adenosine Triphosphatases↗
PubMed1992

Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle.

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.

Animals↗
PubMed1992

Galactosemia: evaluation with MR imaging.

The cerebral findings at magnetic resonance imaging in 67 transferase-deficient galactosemic patients (36 female, 31 male; median age, 10 years) are reported. Twenty-two patients had mild cerebral atrophy, eight had cerebellar atrophy, and 11 had multiple small hyperintense lesions in the cerebral white matter on T2-weighted images. The classic galactosemic patients (those without measurable transferase activity) older than 1 year of age did not show the normal dropoff in peripheral white matter signal intensity on intermediate- and T2-weighted images. The authors postulate that this abnormal signal intensity is due to altered myelin formation secondary to the inability to make sufficient and/or normal galactocerebroside.

Adolescent↗
PubMed1992

The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy.

The mdx mouse has been used for the development of cellular and gene therapies for Duchenne muscular dystrophy. The relatively frequent occurrence of dystrophin-positive muscle cells called revertants has hampered these efforts by interfering with data interpretation. The mdx4cv and mdx5cv dystrophin mouse mutants have approximately 10-fold fewer revertants than the mdx mutant at both 2 and 6 mo. The mdx3cv dystrophin mouse mutant may be a useful model for some types of human dystrophin deficiencies in which the levels of dystrophin are low but not completely absent.

Animals↗
PubMed1992

Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.

3T3 Cells↗
PubMed1991

High-efficiency protein synthesis from T7 RNA polymerase transcripts in 3T3 fibroblasts.

After NIH3T3 cells constitutively expressing T7 RNA polymerase were transfected (+ Ca.phosphate) with a circular DNA containing the firefly luciferase(Luc)-encoding gene (luc) 3' to the encephalomyocarditis (EMC) virus 5'-untranslated sequence and T7 promoter, Luc protein comprising approx. 20% of total cellular protein was obtained. After similar transfection of an analogous construct containing the lacZ gene into the same cell line, at least 50% of the cells produced beta-galactosidase. Fibroblasts lipofected with uncapped RNA transcripts containing EMC sequence expressed the reporter genes as efficiently as capped transcripts. A novel approach was used to generate RNA transcripts containing poly(A) at its very 3' end. RNA from a luc vector with a poly(A) sequence at the very 3' end produced 20-fold more Luc than the RNA from the same vector with an additional 3' nonpoly(A) sequence. These results suggest that this T7 RNA polymerase expression system will be useful for the efficient production of proteins in mammalian cells.

3T3 Cells↗
PubMed1991

Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs.

Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.

Animals↗