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 gene-trap vector, pWH14, has been developed to tag genes expressed in embryonic stem (ES) cells of the mouse. The approach relies on the ability of the endogenous promoter to drive promoterless neo-IRES-lacZ construct producing a dicistronic mRNA consisting of the neomycin-resistance (neo) gene and the beta-galactosidase gene sequence. The neo gene produces a chimeric protein with the truncated product of the tagged gene and serves as a selectable marker for an insertion into an expressed gene. The internal ribosome entry site (IRES) sequence from murine encephalomyocarditis virus allows the translation of the second cistron, lacZ, to produce beta-galactosidase that can be used as a reporter for the expression of the tagged gene. The pWH14 vector was introduced into ES cells by electroporation, and the cells were selected for G418-resistance. About 50% of the G418-resistant colonies were stained positive for the beta-galactosidase activity. Southern analysis showed that each clone had one or more vector sequences integrated. Northern blot analysis of the clones positive for beta-galactosidase indicated that the fused RNAs containing the neo and the beta-gal genes were derived from the endogenous promoters of the tagged genes. Seven clones were chosen and injected into blastocysts, and chimeras were obtained. Two of the gene-trap insertions (wh14.1 and wh14.3) were transmitted through germ-line. In these two lines, the pattern of lacZ expression was restricted to early stages of embryos. This gene-trap vector may provide a means for tagging and studying the active genes in vivo in early embryogenesis.
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PMID:Dicistronic tagging of genes active in embryonic stem cells of mice. 933 94

Nuclear-targeted beta-galactosidase (beta-gal) is increasingly used as a genetic cell marker in vitro and in vivo. Nuclear sequestration concentrates beta-gal and permits sensitive identification of expressing cells and/or tissues without obscuring the cytoplasmic detail necessary for analysis of cell phenotype. Here, we report the construction and testing of a nuclear-targeted version of the beta geo fusion protein that combines nuclear localization with the ability to select expressing cells with the drug G418. This new marker gene functions efficiently in retroviral vectors and will be useful in identification and isolation of cells transfected in vitro and cells expressing transgenic or gene-targeted constructs in vivo.
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PMID:n beta geo, a combined selection and reporter gene for retroviral and transgenic studies. 934 94

Two delta-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first vector was a traditional construct containing only one delta sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low. The second carried two delta sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-galactosidase levels exceeded those obtained with the 2 mu-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single delta vector.
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PMID:Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae. 935 77

We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5-6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of beta-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, beta-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.
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PMID:Osteoprogenitor cells as targets for ex vivo gene transfer. 944 86

Phosphorylation and dephosphorylation of the retinoblastoma gene product (pRB) are recognized as necessary events in the cell cycle progression. To study the role of pRB in regulation of cell proliferation, the stable cell lines with constitutive expression of the exogenous RB gene can be employed. In order to obtain such cell lines in this work C3H10T1/2 mouse fibroblasts were infected with defective retrovirus encompassing the RB and Neo gene conferring resistance to geniticine (G418). The pRB production and its phosphorylation pattern were analyzed by immunoblotting in cell lysates considering well known data on correlation between pRB phosphorylation pattern and its electrophoretic mobility. Cell lines subjected to G418 selection with the following cloning procedure were identical to the control cells expressing beta-galactosidase, when compared for pRB production and phosphorylation in the cell cycle stages characterized by hyperphosphorylated pRB. However, cells of the experimental cell lines hypophosphorylated pRB much faster and accumulated much more underphosphorylated protein compared to the control cell lines. The doubling time of the cells was not affected either by changes in the pRB phosphorylation pattern or by its overproduction during separate cell cycle stages. These results suggest that maintaining of the physiological level of pRB phosphorylation in cycling cells is strictly controlled and is considered to be a more important condition of the cell cycle progression than pRB dephosphorylation.
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PMID:[Growth of stable clones of mouse fibroblast cell line C3H10T1/2 expressing the human retinoblastoma gene product]. 961 Apr 79

The interaction between plasma sex hormone-binding globulin (SHBG) and its receptor (SHBG-R) inhibits estradiol-induced proliferation of MCF-7 cells (human estrogen-dependent breast cancer) through cAMP and PKA. Thus, SHBG can modulate estradiol action in breast cancer, but the implications of this require a more detailed knowledge of the SHBG-R. To this end, we have transfected MCF-7 cells with an expression vector carrying the human SHBG cDNA (S-MCF-7) and studied the effects of this on both SHBG-R binding and cell proliferation. Control cells were parental MCF-7 (P-MCF-7) and MCF-7 cells transfected with the beta-galactosidase gene (B-MCF-7). Transfections were mediated by lipofectin followed by selection of transfected cells with G418. The amounts of SHBG in culture medium were evaluated by IRMA assay, with only S-MCF-7 cells shown to secrete SHBG; SHBG-R levels were evaluated by tracer binding technique. In P-MCF-7 and B-MCF-7 cells, SHBG-R was detectable as a two-binding site receptor, but no binding of SHBG was observed in S-MCF-7 cells. Proliferation of cells treated with estradiol was evaluated by [3H]thymidine incorporation in the three cell lines and in cells pretreated with SHBG (1 nM) purified from human serum or with conditioned medium from S-MCF-7 cells (medium S). In all three lines, cell proliferation increased after estradiol treatment. Preincubation with purified SHBG was effective in reducing estrogen-induced cell proliferation to basal levels in P-MCF-7 and B-MCF-7 but not in S-MCF-7 cells. The estradiol effect was also inhibited in P-MCF-7 cells treated with medium S. In conclusion, 1) SHBG inhibits estradiol-induced proliferation in cells containing a functional SHBG-R, whereas it has no detectable effect in cells in which the SHBG-R is either absent or not available to bind SHBG; and 2) S-MCF-7 cells are insensitive to SHBG (locally produced or exogenous) because their SHBG-R is occupied by SHBG.
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PMID:Control of the membrane sex hormone-binding globulin-receptor (SHBG-R) in MCF-7 cells: effect of locally produced SHBG. 961 86

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced. However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.
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PMID:Retroviral-mediated marker gene transfer in hematopoiesis-supportive marrow stromal cells. 962 Dec 56

Direct transfer of new genetic information to keratinocytes in epidermis may prove effective in treating certain genodermatoses; however, current methods for in vivo gene transfer to skin do not lead to persistence of the transgene. The goal of this study was to explore direct gene transfer using retrovirus-mediated transduction. Retroviral vectors integrate a DNA copy of their genome into the host chromosome and therefore have the potential to effect a permanent gene therapy. To facilitate development of methods for in vivo transduction with retroviral vectors, a porcine skin organ culture model was constructed in which the denuded surface was repopulated with replicating keratinocytes from hair follicles and epidermal remnants. In situ transduction was carried out by topical application of two retrovirus vectors, MFGlacZ (10(7) blue forming units per ml) and LZRN pseudotyped with the G protein of vesicular stomatitis virus (VSV) (10(9) colony forming units per ml), each encoding the beta-galactosidase reporter gene and the latter encoding the neomycin phosphotransferase selectable gene. Beta-galactosidase expressing cells were observed more frequently with LZRN than with MFGlacZ; however, transduction efficiency remained low in both instances. At equivalent titers, the VSV-G pseudotyped retroviral vector was shown to transduce porcine keratinocytes more efficiently than a similar vector with the amphotropic envelope. The number of beta-gal+ cells in organ culture could be increased by selection of LZRN-transduced cells in situ with G418. To achieve transduction of epidermis in vivo, these studies point out the importance of high titer retroviral vectors, pseudotyping with VSV-G protein, and in situ selection.
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PMID:Retrovirus-mediated transduction of porcine keratinocytes in organ culture. 974 Feb 46

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.
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PMID:High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. 976 32

Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or beta-galactosidase (beta-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells.
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PMID:Targeting bacteriophage to mammalian cell surface receptors for gene delivery. 982 29


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