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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.
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PMID:Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial. 1081 36

We have initiated a phase I/II clinical trial, involving the use of herpes simplex thymidine kinase gene (HS-tk)-expressing donor primary T cells, in order to modulate the graft-versus-host disease (GvHD) occurring after allogeneic hematopoietic stem cell transplantation. The preparation of gene-modified T cells (TkTCs) required a 12-day ex vivo culture comprising an initial OKT3 and IL-2 stimulation, a retrovirus-mediated transduction, and a 7-day selection step in the presence of G418 and IL-2. The low transduction efficiency as well as the culture conditions may significantly alter the diversity of the T cell repertoire. We therefore examined the T cell repertoire of HS-tk-expressing T cell samples from 11 different donors by the Immunoscope method. This method analyzes the hypervariable region of the T cell receptor beta chain (TCRBV) by amplifying the complementarity-determining region 3 (CDR3) and determining size diversity. In all examined samples (four of which were infused into patients), all TCRBV subfamilies were represented with, however, a significant skewing within a minority of subfamilies. Kinetic studies demonstrated that this skewing appeared between day 7 and day 12, with dates of appearance variable from one subfamily to another. In addition, the repertoire analysis of two different culture products, harvested and produced at different times from the same donors, suggested that some repertoire abnormalities could be donor specific. Quantitative analysis revealed no major modifications in gene usage, even in skewed TCRBV subfamilies, with a few clonal expansions concerning a limited number of TCRBV subfamilies. Importantly, identical abnormalities were found in control cells grown in parallel under similar conditions but not transduced or selected, thus demonstrating that these abnormalities were not related to the transduction or the selection process, but rather to the ex vivo culture. The initial stimulus used for T cell activation is a major source of TCRBV perturbation, since replacing the OKT3 + IL-2 stimulus by CD3 + CD28 monoclonal antibody-coated beads prevented the occurrence of alterations. Overall, the HS-tk-expressing T cells used in our clinical trial exhibit limited TCR repertoire skewing that is not due to the transduction/selection procedure. However, future T cell gene transfer protocols for clinical trials should be designed to take into account or possibly prevent such T cell repertoire alterations.
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PMID:Retrovirus-mediated gene transfer in primary T lymphocytes: influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire. 1083 17

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.
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PMID:T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection. 1088 24

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.
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PMID:Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir. 1088 25

Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.
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PMID:Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells. 1474 37

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.
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PMID:Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish. 1507 74

We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.
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PMID:Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1. 1508 8

We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."
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PMID:A role for DNA mismatch repair protein Msh2 in error-prone double-strand-break repair in mammalian chromosomes. 1578 95

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.
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PMID:[The ht-PAm cDNA knock-in the goat beta-casein gene locus]. 1597 6

Gene targeting is a more powerful method to produce mammary gland bioreactor and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe an efficient and reproducible gene targeting in goat mammary epithelium cell to place the GFP and neo at the beta-casein locus. The transgenic goat would be produced by nuclear transfer. To construct the gene targeting vector pGBC-GFP-neo, the milk goat beta-casein genomic DNA sequence for homologous arms was cloned first. The left arm was 2.1 kb fragment including goat beta-casein gene exon1 and part of exon 2, and the right arm was 5.1 kb fragment including beta-casein gene from exon 7 to 3'-flanking sequence. The bacterial neomycin (neo) gene as positive selection marker gene, with the promoter-trap GFP, was placed between two loxPs. The thymidine kinase (tk) as negative selection marker gene was just outside the right or left arms. Goat mammary epithelium cells were cultured to sub-confluence about 90% and transfected with linear pGBC-GFP-neo using Lipefectamin-2000. These transfected cells were cultured in collagen-coated 96-wellplate for 24 h without selection, then added the drug G418(600 microg/mL) and GANC(2 micromol/L). After nine days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate; 51 clones were selected; 17 clones were tested by GFP expression using promoter-trap strategy; only four clones grow well. After PCR confirmation the four targeting cell clones homologous recombination were used as the donor cell for nuclear transfer. 59.5% cloned embryos could develop up. Some could develop to morula or blastocyst in vitro.
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PMID:[High-efficient gene targeting of goat mammary epithelium cell by the multi-selection mechanism]. 1601 Oct 27


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