Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A large cassette, 4.6 x 10(3) bases (4.6 kb) in length, containing an inducible expression system (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene) and a bacterial neomycin-resistance gene (neo) has been cloned into the noncoding region of a GAL1-regulated Ty1 retrotransposon. Galactose was used to induce retrotransposition in Saccharomyces cerevisiae, and cells containing integrations were selected by resistance to the aminoglycoside G418. Integrations of neo and CUP1p-lacZ were verified, and beta-galactosidase activity was confirmed. Analysis via Southern blots demonstrated integrations at various chromosomal locations, and the number of insertions obtained ranged from one to five after three rounds of induction. Therefore, the packaging limit of Ty1 virus-like particles for RNA is at least 10.3 kb and Ty1 can transpose foreign genes as large as 4.6 kb, demonstrating the practical application of Ty1 for the insertion of large regulated expression cassettes.
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PMID:Application of Ty1 for cloned gene insertion: amplification of a large regulated expression cassette in Saccharomyces cerevisiae. 870 32

The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding sequences for a beta-galactosidase-neomycin phosphotransferase hybrid protein. Integrated proviruses, which disrupted expressed cellular genes, were selected in the presence of G418. ES clones expressing regulated beta geo fusion genes were identified by changes in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining after in vitro differentiation. Thirty-one of 191 clones tested (16%) exhibited regulated expression of beta geo protein. Seven genes disrupted by U3 beta geo were passed into the germline, and expression of the beta geo fusion genes was analyzed in vivo, including inserts disrupting the Eck and REX-1 genes. In each case, genes trapped in cultured ES cells were expressed in the inner cell mass of preimplantation embryos, and changes in lacZ expression during in vitro differentiation were also observed during early development. Thus, cultured ES cells maintain, to a considerable extent, the transcriptional specificity of the pluripotent cells of the preimplantation embryo. As a consequence, in vitro screens utilizing gene traps provide a rapid and accurate means to identify and disrupt developmentally regulated genes.
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PMID:Transcriptional specificity of the pluripotent embryonic stem cell. 889 43

The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.
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PMID:Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system. 898 90

Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as beta-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (approximately 50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex.
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PMID:Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery. 903 23

Replication-defective, highly purified retroviral vectors (Retrovector), at titers of 10(8) colony forming units/mL, were prepared that conferred either beta-galactosidase or herpes simplex thymidine kinase (HSV-TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (GCV). The mean frequency of in situ transduction, measured by flow cytometry of single-cell tumor suspensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessments were made by staining of beta-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK gliosarcoma tumors treated with GCV were eradicated in approximately 53% of the animals (10/19) at day 26, however, 89% (17/19) histologically showed < 1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV-TK vector followed by GCV treatment showed that 29% (4/14) had no tumor; 50% (7/14) had < 1% tumor volume. Regression of tumors proceeded over the time since the complete rate was increased at day 60. Neither HSV-TK vector particles nor GCV alone altered the histological profile of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocytes infiltrated the tumors of animals treated with both. In cured animals, the former tumor bed contained cell debris, immune cells, and fibroblasts and was without damage to adjacent brain. The efficacy of suicide gene therapy for rat gliosarcoma using highly purified virion vectors approaches that of packaging cell lines.
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PMID:Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors. 908 Jan 21

The herpes simplex virus type 1 (HSV-1) mutant in 1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in 1820. In almost all cell types tested, in 1820 initiated infection less efficiently than in 1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in 1820 was less impaired than in 1814. A rescuant of in 1820 at the VP16 locus, in 1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In 1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in 1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1 tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in 1820 to produce in 1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in 1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative of in 1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in 1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in 1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors.
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PMID:Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression. 912 65

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.
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PMID:In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures. 917 33

We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable lacZ/neo fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of beta-galactosidase (beta-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express beta-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by lacZ/neo in the hybrid gene trap transcript. This approach, which we term "in vitro preselection," is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.
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PMID:In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse. 918 83

We have previously described a method for the derivation of long term cultures of undifferentiated myoblasts from the skeletal muscle of dystrophic (mdx) mice (J. Smith and P. N. Schofield, Exp. Cell Res., 210: 86-93, 1994). We now show that a clonal mdx-derived skeletal muscle cell line labeled with a retrovirus conferring beta-galactosidase activity and G418 resistance (PD50A) is capable of incorporation into mdx skeletal muscle myofibers for up to 14 months with no incidence of tumor formation. After a lag period of 5 days, injected PD50A cells disperse throughout the injected tibialis anterior muscle and take up satellite cell positions on the perimeter of myofiber bundles. PD50A cells begin to incorporate into fused muscle syncitium as early as 8 weeks after injection and persist for at least 14 months. We have rederived myoblasts expressing beta-galactosidase from PD50A-injected muscles 12 months after injection, demonstrating that a reserve of mononuclear proliferation-competent PD50A cells are present in host muscle up to a year after their original introduction. These data support the contention that myoblasts derived by this culture method are functionally representative of a class of skeletal muscle "stem cells" and thus have potential both as agents for cellular therapy of intransigent diseases such as Duchenne muscular dystrophy as well as being a useful tool for the further investigation of normal muscle development.
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PMID:Stable integration of an mdx skeletal muscle cell line into dystrophic (mdx) skeletal muscle: evidence for stem cell status. 926 2

A new retroviral vector has been constructed that expresses genes encoding three different activities from a single transcript. This feature has been exploited to enable the efficient marking and selection of cells that express a gene of interest. The marker gene lacZ, encoding beta-galactosidase, and neo, encoding neomycin phosphotransferase, for selection by the antibiotic G418, are expressed as a fusion, beta Geo. The expression of beta Geo is coordinated with expression of a gene of interest at the mRNA level using an Internal Ribosome Entry Site (IRES) from the Encephalomyocarditis Virus (EMCV). The IRES promotes cap-independent initiation of translation therefore two reading frames can be translated from a single transcript. In vitro, the vector has been shown to confer beta-galactosidase activity, transformation by v-src and resistance to G418, following infection of cells. To show that the retrovirus was able to mark infected cells in vivo, cells infected with the retrovirus were transplanted into mouse mammary gland where they grew and were successfully located by staining for beta-galactosidase over 2 months after transplantation.
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PMID:A new retroviral vector, CA1, to identify and select for cells expressing an inserted gene in vitro and in vivo. 932 57


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