Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.
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PMID:Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus. 204 98

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.
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PMID:Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus. 274 40

The herpes simplex virus thymidine kinase (HSV-tk) gene conferring ganciclovir (GCV)-specific sensitivity to transduced cells might control Graft-versus-Leukemia (GvL)/Graft-versus-Host Disease (GvHD). Human T lymphocytes were engineered with an LSN-tk retroviral vector encoding tk and neomycin resistance (NeoR) genes. A total of 80 x 10(6) tk(+) lymphocytes were injected intraperitoneally in NOD-SCID mice. Engraftment was evaluated by human CD45(+)/CD3(+) cytofluorimetric analysis and NeoR-based polymerase chain reaction (PCR) on peripheral blood, bone marrow, liver, thymus, and spleen on day +5. After 14 days, GCV (10 mg/kg daily) cytofluorimetric analysis and PCR were repeated (day +19). Immunohistological studies with anti-CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti-alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45(+)/CD3(+) cells were engrafted in all tissues on day +5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes "homed" to the white pulp T-cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV (day +19), cytofluorimetry and immunohistology showed very few CD3(+) cells. PCR identified the transgene in 22% tissue samples (positive only in thymus and spleen). GvHD did not occur in any animal. These data demonstrate elevated doses of human-transduced CD3(+) cells engraft in NOD/SCID mice; after GCV, very few CD3(+) cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day +19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk-transduced T lymphocytes.
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PMID:Homing and survival of thymidine kinase-transduced human T cells in NOD/SCID mice. 1218 25

To develop a trial with lymphocyte suicide gene therapy in patients with hematological malignancies, we transduced human T lymphocytes with a retroviral vector (LSN-tk) encoding the herpes simplex virus thymidine kinase (tk) and the neomycin resistance (NeoR) genes. Precise quantification of gene transfer is crucial for any gene therapy protocol, but methods using semiquantitative PCR are inaccurate and subject to variations. Real-time quantitative PCR could be a valid alternative. A TaqMan probe was designed to hybridize with the NeoR gene. The PCR product is 64 nucleotides long and readily quantified by TaqMan probe binding. The analysis was performed soon after transduction and repeated after the selection procedure. This method was more accurate, reproducible, and sensitive than the semiquantitative PCR assay. Accuracy was the same whether the analysis was performed at the highest rate or at the lowest rate of transduction. Additionally we used real-time PCR to monitor the kinetics of enrichment of the transduced cells over the selection time and showed how 7 days of selection are needed. This study precisely quantified the percentages of cells transduced by the retroviral vector and could have major implications in gene therapy studies.
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PMID:NeoR-based transduced T lymphocytes detected by real-time quantitative polymerase chain reaction. 1266 39