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

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.
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PMID:DNA sequence responsible for the amplification of adjacent genes. 367 95

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.
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PMID:Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood. 751 1

We have used an insertion vector-based approach to target the G(i2) alpha gene in AB-1 embryonic stem cells. 105 bp located 0.8-0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineered Not I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained ('hit' step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy,2-fluoro-beta-D-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination ('run' step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified 'hit and run' strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G(i2) alpha allele is also demonstrated.
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PMID:Disruption of the G(i2) alpha locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair. 826 81

Recombinant human adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) promoter (vTK-Neo), the murine colony-stimulating factor-1 (CSF-1) promoter (vCSF1-Neo) or the CSF-1 promoter plus an upstream human erythroid cell-specific enhancer, HS-2 (vHS2-CSF1-Neo). Recombinant virions were used to infect low-density murine primary bone marrow cells. In hematopoietic progenitor cell assays initiated with cells infected with these recombinant virions, myeloid as well as erythroid cell colonies resistant to the drug G418, a neomycin analogue, were readily obtained, indicating that the murine hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions and that the transduced neo gene was functionally active in these cells. Whereas only approximately 10% of the colony-forming unit-granulocyte/macrophage (CFU-GM) colonies cloned from mock-infected cells survived the G418-selection at a final active concentration of 250 micrograms/mL of the drug, the extent of the CFU-GM colony formation initiated with the recombinant AAV-Neo virions was as follows: 15% with vTK-Neo, 22% with vCSF1-Neo and 49% with vHS2-CSF1-Neo. In addition, only 14% of the burst-forming unit-erythroid (BFU-E) colonies from mock-infected cells were resistant to G418, whereas 82% of the BFU-E colonies initiated with cells infected with vHS2-CSF1-Neo virions survived the drug selection, suggesting that a human erythroid cell-specific enhancer was able to potentiate expression of the transduced neoR gene from a murine promoter. Individual CFU-GM and BFU-E colonies from mock-infected or recombinant AAV-Neo virus-infected cultures were subjected to polymerase chain reaction (PCR) analysis using a neo-specific synthetic oligonucleotide primer-pair. A 276 bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was detected only in colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest the feasibility of using the AAV-based vector system in an animal model as a prelude to evaluating its safety and efficacy in human gene therapy.
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PMID:Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. 839 71

Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic Gi2 alpha sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the NcoI site of exon 3. G418RFIAUR clones corresponding to ca. 4 x 10(8) ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418RFIAUS), which was lost--together with the plasmid and the TK sequences--by intrachromosomal recombination (Run step; G418RFIAUR). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.
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PMID:Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors. 845 May 7

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.
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PMID:Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. 904 94

We have previously shown that transfer of a mutated dihydrofolate reductase (DHFR) confers resistance to methotrexate (MTX) to infected cells. We report herein the construction of a retrovirus vector, DC/SV6S31tk, which carries the herpes simplex virus thymidine kinase gene (HSVtk) as well as the mutated Serine 31 DHFR (S31) cDNA. 3T3 cells infected with DC/SV6S31tk are more resistant to MTX than cells infected with DC/SV6S31, which carries the S31 and Neo gene. In DC/SV6S31tk-infected cells, a fraction of cells (20-40%) were more resistant to MTX compared with DC/SV6S31-infected cells, and these cells survived a 5-day exposure to 200 microM of MTX. The mechanism of this augmented resistance is attributed to the salvage of thymidine by HSVtk, as the augmentation is reversed when dialyzed serum is used for cytotoxicity assays. The cells that survive high-dose MTX selection have high levels of expression of S31 DHFR and HSVtk, although copy numbers of the proviral sequences do not increase significantly. Transduction of cells with the DC/SV6S31tk vector also sensitizes cells to ganciclovir (GCV). Co-expression of a metabolically related gene in a retroviral vector to potentiate the resistance imparted by a drug resistance gene may be useful for gene therapy for cancer patients.
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PMID:Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene. 923 Oct 73

Vaccination with cytokine-transduced tumor cells represents a potentially important approach to the treatment of central nervous system tumors. We have recently demonstrated the therapeutic efficacy of tumor cell vaccines expressing the murine interleukin 4 (IL-4) and the herpes simplex virus-thymidine kinase in a rat brain tumor model in which nonirradiated vaccine cells can be eliminated by the subsequent administration of ganciclovir. In this report, we demonstrate the construction and characterization of a retroviral vector that encodes human IL-4, neomycin phosphotransferase, and herpes simplex virus-thymidine kinase genes for use in human clinical trials. An MFG-based retroviral vector was used to generate the recombinant retrovirus, TFG-hIL4-Neo-Tk, in which a long terminal repeat-driven polycistronic transcript encodes three cDNAs that are linked and coexpressed using two intervening internal ribosome entry site fragments from the encephalomyocarditis virus. The amphotropic retroviral vector TFG-hIL4-Neo-Tk was then used to infect human primary glioma cultures and skin-derived fibroblasts. After infection and G418 selection, cells produced 89-131 ng/10(6) cells/48 hours of human IL-4, which was determined to be biologically active. Transduced glioma cells were highly sensitive to the cytotoxic effect of ganciclovir. These data demonstrate the suitability of the TFG-hIL4-Neo-Tk vector for therapeutic studies of cytokine-transduced autologous tumor vaccination in patients with malignant gliomas.
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PMID:Characterization and transduction of a retroviral vector encoding human interleukin-4 and herpes simplex virus-thymidine kinase for glioma tumor vaccine therapy. 1076 55

The in vivo gene delivery of E. coli cytosine deaminase (cd) cDNA and systemic 5-fluorocytosine (5-FC) administration have been studied extensively because of their clinical relevance to cancer gene therapy. This approach has the potent advantage of a stronger bystander effect compared to the previous thymidine kinase suicide gene system of the herpes simplex virus. However, 5-fluorouracil (5-FU), an active metabolite in cd with 5-FC therapy, is not always effective for every type of tumor since the enzymes responsible for further drug metabolism vary significantly in each tissue. In this study, we aimed to increase the sensitivity of 5-FU by transduction of thymidine phosphorylase (dThdPase) cDNA into brain tumor cells. After retroviral transfer of the cDNA, we obtained 9L murine gliosarcoma cells showing stable expression of the target enzyme (9L-dThdPase). The growth of the cells was identical to wild type (9L-WT) or control-vector transfected (9L-Neo) cells in vitro. Sensitivity to 5-FU was increased in 9L-dThdPase cells. After the adenoviral delivery of cytosine deaminase gene into these cells, 9L-dThdPase cells also demonstrated an increased sensitivity to 5-FC. Moreover, we showed that transduction of dThdPase cDNA prolongs the survival of animals bearing intracerebral tumors after experimental in vivo cytosine deaminase gene therapy. These results suggest that transduction of thymidine phosphorylase may be a beneficial approach to increasing the efficacy of cd/5-FC suicide gene therapy in certain types of tumor.
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PMID:Transduction of thymidine phosphorylase cDNA facilitates efficacy of cytosine deaminase/5-FC gene therapy for malignant brain tumor. 1172 81

Reportedly, in human immunodeficiency virus type 1 (HIV) vectors, insertion of central polypurine tract (cPPT) increased expression of transgenes for a short period. To test this for a stable condition, we constructed a series of vectors carrying a Neo(r) gene as a stable marker driven by a synthetic thymidine kinase (hTK) promoter. Transduction efficiency was increased in about 2-fold and decreased in about 8-fold by insertion of the reported 178bp and our 282bp cPPTs, respectively. PCR analyses revealed that insertion of 282bp cPPT, but not 178bp cPPT, impaired integration, although it did not deteriorate nuclear transport much. Furthermore, we found that insertion of 282bp cPPT between hTK promoter and an upstream LTR sequence reduced reporter gene activity in about 5-fold. This inhibitory effect of 282bp cPPT may partly account for the observed decrease in transduction efficiency. We suggest that actual effect of cPPT insertion should be examined in each HIV vector.
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PMID:Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1. 1261 60


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