To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism

To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genetic Therapy ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; Humans ; K562 Cells ; Thymidine Kinase ; genetics

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo investigate enhancement of the bystander effect by tanshinone IIA (Tan) in HSV-tK/GCV system and the correlation with expression of connexin 43 mRNA.

METHODSThe cytotoxic effect in HSV-tK/GCV in cervical carcinoma cell line ME180 (ME) and ME/TK was examined by MTT assays. Cx43 mRNA expression was detected by fluor-quantitative RT-PCR.

RESULTSTan markedly increased sensitivity of ME/TK cells for GCV in HSV-tK/GCV system. In the presence of 2 micro g/ml GCV, compared with the absence of Tan (0 mol/L), an obvious decrease in survival rate was seen at any given mixture of ME and ME/TK cells exposed to 1.3 x 10(-9) mol/L Tan. Statistics showed significant difference (P < 0.05). However, enhancement of bystander mediated cell killing occurred only in the range of Tan concentrations used (1.3 x 10(-8), 1.3 x 10(-9) mol/L). RT-PCR showed that the ratio of relative copy number of Cx43 mRNA increased by 8.83 and 8.47-fold in ME cells exposed to 1.3 x 10(-8) and 1.3 x 10(-9) mol/L Tan, respectively.

CONCLUSIONFor the first time we report that in cervical carcinoma ME180 cell line, Tan possesses a remarkable enhancing role on the bystander effect in the HSV-tK/GCV system. It is associated with up-regulation of Cx43 mRNA expression.

Antineoplastic Agents, Phytogenic ; pharmacology ; Bystander Effect ; Cell Line, Tumor ; Cell Survival ; drug effects ; Connexin 43 ; genetics ; Diterpenes, Abietane ; Female ; Ganciclovir ; pharmacology ; Genetic Therapy ; Humans ; Phenanthrenes ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Uterine Cervical Neoplasms ; therapy

OBJECTIVETo evaluate the lethal effect of adenovirus-mediated HSV-TK-ganciclovir (GCV) gene therapy in combination with hydroxycamptothecin (HCPT) on hunman bladder carcinoma cell line T-24 cells.

METHODSHuman bladder carcinoma cell line T-24 was transfected with adenovirus expression vector containing TK gene and green fluorescent protein (GFP) gene, and the transfection efficiency was observed and TK expression detected by PCR. After successful cell transfection indicated by GFP expression, GCV and hydroxycamptothecin are respectively added into the cell culture with normal T-24 cells serving as the blank control group. The growth inhibition rate of hunman bladder carcinoma cells in response to HCPT treatment for 72 h and the cell survival rate of 24 h, 48 h and 72 h after transfection with different protocols were observed by MTT assay. The apoptosis of the cells treated with GCV (0.5 mg/ml)+HCPT (10 mg/L) for 4 h was observed by flow cytometry.

RESULTSThe cell inhibition rate increased gradually with increment of HCPT concentration, from 14% at HCPT concentration of 0.01 mg/L to 60% at 50 mg/L, but for a concentration above 100 mg/L, the inhibition rate did not exhibit further increase (P=0.216). GCV alone and GCV in combination with HCPT both resulted in significantly decreased survival rate of human bladder carcinoma cells (P=0.00), and the killing efficiency of the cells by GCV+HCPT protocol increased obviously with increment of HCPT concentration and prolongation of the action time. The cells treated with 0.5 mg/ml GCV alone for 72 h retained a cell survival rate of 34.6%, which was lowered to only 8.07% with combined treatment with GCV (0.5mg/ml) and HCPT (10 mg/L). Typical apoptotic peak before M1 phase of the cells appeared 4 h after treatment with GCV+10 mg/ml HCPT, which resulted in a apoptosis rate of 52.93%.

CONCLUSIONHSV-TK/GCV in combination with HCPT can enhance the lethal effect of suicide gene therapy against human bladder carcinoma cells and effectively induce apoptosis of the cells.

Adenoviridae ; genetics ; Apoptosis ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Thymidine Kinase ; pharmacology ; Transfection ; Urinary Bladder Neoplasms ; therapy

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.
Antiviral Agents ; pharmacology ; Bystander Effect ; Cell Line, Tumor ; Connexin 43 ; biosynthesis ; genetics ; Female ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; Humans ; Ovarian Neoplasms ; metabolism ; therapy ; Pregnancy ; Simplexvirus ; genetics ; Thymidine Kinase ; genetics ; Tretinoin ; pharmacology

OBJECTIVETo study the killing effect of human herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system combined with allitride and the possible apoptosis mechanism in BIU87 cells.

METHODSThe cytotoxicity after combination were estimated by theamine blue tetrazolium bromide (MTT). The morphological changes were observed with inverted microscope and in-situ cell apoptosis detection kit. Changes of apoptosis rate and cell cycle were assessed by flow cytometry. B-cell lymphoma-2 (bcl-2), bax, caspase-3 (cysteine aspartate specific proteinase) mRNA changes were detected by reverse transcriptase polymerase chain reaction, and caspase-3 activity was estimated with colorimetry.

RESULTSFor combination group, the cell killing rate was raised to 72.50% to compare with 35.00% of GCV and 37.00% of allitride separately and there was a synergistic effect between these two drugs. The cell apoptosis was induced in all three groups and for the combination group the time of S-phase and G(2)-phase arrest were earlier than other two groups. Both drugs could inhibit the expression of bcl-2 and promote the expression and activity of caspase-3.

CONCLUSIONSThe combination of HSV-TK/GCV system with allitride can inhibit the proliferation of BIU87 cells congenerously through apoptosis, which may be correlated with S- and G(2)-phase arrest, down-regulation of bcl-2 and increased caspase-3 expression and its activity.

Apoptosis ; drug effects ; Drug Synergism ; Ganciclovir ; pharmacology ; Genetic Therapy ; Herpesvirus 1, Human ; enzymology ; genetics ; Humans ; In Vitro Techniques ; Sulfinic Acids ; pharmacology ; Thymidine Kinase ; genetics ; Transfection ; Urinary Bladder Neoplasms ; pathology ; therapy

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Recombination, Genetic ; Retroviridae ; genetics

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

OBJECTIVETo explore the killing effect of the mutant herpes simplex virus thymidine kinase (HSV-sr39tk) and its wild-type (HSV-tk) mediated by lentiviral vector on T lymphocytes in vitro and compare T cell survival rate after GCV or ACV treatment.

METHODSThe three-plasmid lentiviral vector system including packaging plasmid DeltaNRF, envelope plasmid VSV-G and vector plasmid (pTK151 + HSV-sr39tk or pTK151 + HSV-tk) were cotransfected into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations.

RESULTSThe three plasmids were effectively cotransfected and a high titre of lentivirus was obtained (2 x 10(6) IU/ml). 39tk(+) T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 micromol/L to 10 micromol/L. The T cell survival rates in GCV group declined from (96.04 +/- 3.23)% to (36.76 +/- 4.38)% while in ACV group from (97.31 +/- 4.61)% to (43.75 +/- 8.99)%. However, when GCV/ACV concentrations were more than 10 micromol/L, further decline of 39tk(+) T cell survival rates became unobvious. The growth rate of 39tk(+) T cell exposed to GCV or ACV was obviously lower than that in un-transfected T cells (P < 0.05). Tk(+) T cells were sensitive to GCV (P < 0.05), but not to ACV (P > 0.05). There was a significant difference in killing effects between 39tk(+) T cell + GCV group and tk(+) T cell + GCV group (P < 0.05).

CONCLUSIONThe lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.

Acyclovir ; pharmacology ; Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Ganciclovir ; pharmacology ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; T-Lymphocytes ; cytology ; drug effects ; Thymidine Kinase ; genetics ; Transfection