Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used the 1.1 kilobases of the 5' upstream region of the platelet factor four (PF4) gene coupled to the prokaryotic beta-galactosidase gene to generate two lines of transgenic mice that express this construct. Studies of blood, bone marrow, spleen, and thymus reveal that platelets are the only circulating blood cells and megakaryocytes are the only hematopoietic precursor cells that possess the prokaryotic enzyme. The lack of transgene expression in brain, heart, intestine, kidney, liver, lung, and skeletal muscle was established by in situ staining of tissue sections as well as kinetic assay of tissue homogenates. These data suggest that this domain of the PF4 promoter contains most, if not all, of the tissue-specific region of the gene. Unexpectedly, the adrenal gland exhibits approximately 2% of the levels of beta-galactosidase possessed by megakaryocytes and the distribution of the prokaryotic enzyme corresponds to the location of mineralocorticoid-secreting cells. This result implies that either the PF4 gene is transcribed at low levels in specialized adrenal cells or that these specialized endocrine cells possess trans-acting factors similar to those that control the megakaryocyte promoter. The selective high-level expression of transgenes linked to the PF4 promoter should allow us to augment or suppress the in vivo levels of critical components in megakaryocytes and platelets and subsequently ascertain the effects of these modifications.
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PMID:Selective targeting of gene products with the megakaryocyte platelet factor 4 promoter. 189 30

There have been no reports, to date, of successful introduction of foreign DNA into committed megakaryocyte precursor cells. We have successfully infected two megakaryocytic cell lines, one a committed cell line (CHRF-288-11) and the other a bipotential cell line (K562), with a retroviral vector containing the bacterial lacZ gene and a neomycin resistance marker. Modification of standard protocols was required for successful infection of the committed megakaryocyte cell line. Presence of the lacZ transgene was demonstrated at the molecular level by polymerase chain reaction (PCR), and its expression at the mRNA level by reverse transcriptase-PCR. Presence of the bacterial beta-galactosidase was demonstrated by both immunofluorescence and enzyme activity. Treatment of the CHRF-288-11 infected cells with phorbol esters, which induces megakaryocytic differentiation, increases expression of the lacZ transgene. The staining pattern of the lacZ reaction product was perinuclear and punctate in the CHRF-288-11 cells, whereas it was uniform throughout the cytoplasm of the K562 cells, suggesting different sorting mechanisms for bacterial beta-galactosidase in these two cell types. Overall, these results demonstrate the feasibility and provide a method for infecting cultured megakaryocytic cell lines with retroviral of vectors such that a molecular analysis of megakaryocyte differentiation can be accomplished.
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PMID:Retroviral mediated gene transfer in megakaryocytic cell lines. 788 34

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5'-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with -245 bp of 5'-flanking region were more active than constructs with -2 kb of 5'-flanking region in vitro. We created two independent transgenic mouse lines with a -245-bp hPF4/lacZ construct. Cells from these mice were tested for beta-galactosidase (beta-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed beta-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no beta-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The -245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.
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PMID:-245 bp of 5'-flanking region from the human platelet factor 4 gene is sufficient to drive megakaryocyte-specific expression in vivo. 951 30

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.
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PMID:Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells. 1044 49

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.
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PMID:Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression. 1059 53