OBJECTIVE: The transfection efficiencies of gynecologic cancer cell lines were investigated by different mediated transfection methods using recombinant LacZ plasmid (pRcCMVLacZ and pAAVCMVLacZ). METHODS: In this study, the gynecologic cancer cell lines were used CaSki, SiHa (cervical, HPV16+, wild type p53 gene), HeLa, HeLa S3 (cervical, HPV18+, wild type p53 gene), C33A, HT3 (cervical, HPV-, p53 mutant), HckE6/E7 (cervical, HPV16 immortalized keratocyte), PA-1 (ovary, wild type p53), SKOV-3, A2774 (ovary, p53del) and OVCAR-3 (ovary, p53 mutant). The pRcCMVLacZ and pAAVCMVLacZ plasmid transfection were performed by using liposome system such as Ca2+-phosphate, Fugen6(TM), Lipofection(TM), Lipogen(TM) and N-stearyl lactobionamide (N-SLBA) with X-gal staining. The LacZ gene was used the reporter gene for the transfection efficiencies evaluation. RESULTS: Each of cell lines were showed different transfection efficiencies by Ca2+-phosphate, Fugen6(TM), Lipofectin(TM), Lipogen(TM) and N-SLBA. Each of cell were revealed that HeLa S3, HT3 and A2774 were high transfection efficiency using the pRcCMVLacZ by the Lipogen(TM), SiHa, HeLa, QGU, OVCAR-3 and PA-1 were high efficiency using the pAAVCMVLacZ by Lipofectin(TM), CaSki was high efficiency using the pRcCMVLacZ by the Lipogen(TM), A2774 and Cx16.2 were high efficiency using the pRcCMVLacZ by the Lipofectin(TM), SKOV-3 and HkcE6/E7 were high efficiency using pAAVCMVLacZ by the Lipogen(TM). CONCLUSION: As a result, We proved that each of cell lines differed trasnfection efficiencies according to mediated transfection and recombinant LacZ plasmid style. Above all, Lipofectin(TM) mediated transfection was showed high efficiency at the most of cell lines.
Cell Line* ; Genes, Reporter ; Humans* ; Lac Operon* ; Liposomes ; Plasmids* ; Transfection*

Among a number of antigens characterized in M. leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M. leprae. We have previously determined a sequence specific element in the 18 kDa gene of M. leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M. leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M. leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M. bovis BCG or M. smegmatis in front of LacZ gene resulted in normal beta- galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, beta-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the beta-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M. leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.
beta-Galactosidase ; Clone Cells ; Cloning, Organism ; Galactosidases ; Genes, Reporter ; Lac Operon ; Leprosy ; Mycobacterium bovis* ; Mycobacterium leprae ; Repression, Psychology*

Using lactoferrin as the specific ligand, we developed a simplified method for preparation of molecular conjugate for gene delivery. Replacement of column chromatography and dialysis by one step centrifugal filtration (Centricon, cut off size : 30,000), resulted in the rapid purification of bovine lactoferrin/polylysine (bLf/pL) and human lactoferrin/polylysine (hLf/pL) conjugates and easy separation of unconjugated polylysine. The Lf/pL conjugates prepared by this method efficiently transferred the reporter genes, CAT and LacZ gene, to HeLa and hepatic cells. The bLf/pL and hLf/pL conjugates could transfer the reporter genes to various hepatocytes including primary mouse hepatocyte, Hepa 1-6, SK-Hep1 and Chang liver, but not to NIH 3T3 mouse fibroblast cells, indicating that the Lf/pL conjugates conferred hepatocyte-specific gene transfer. The bLf/pL and hLf/pL conjugates prepared in the present study exhibited higher transfection efficiencies for mouse and human hepatocytes than the commercially available transferrin/polylysine (Tf/pL) conjugate.
Animals ; Cats ; Chromatography ; Dialysis ; Fibroblasts ; Filtration ; Genes, Reporter ; Genes, vif* ; Hepatocytes* ; Humans ; Lac Operon ; Lactoferrin* ; Liver ; Mice ; Polylysine ; Transfection

PURPOSE: There are several reasons why retroviruses are useful as vectors for gene therapy. However, retroviral vectors also have some limitations. Research in retroviral-mediated gene transfer has struggled with low titer and transduction efficiency on certain human target cells even with the addition of polycations to enhance transduction. Efficient in vivo gene transfer with retroviral vectors will require the availability of large amounts of vector at titers higher than generally possible by most current methods. Therefore, transduction efficiency of various human cell types with retroviral vector system is very important in human gene therapy. In an effort to test the transduction efficiency of a retrovial vector in the human cancer cell lines, a retroviral vector was infected into various human cancer cell lines. MATERIALS AND METHODS: We generated retrovirus producing cell lines through transfection or infection of amphotropic packaging cell line PA317 with ecotropic retroviruses encoding bacterial lacZ gene. The amphotropic retrovirus vector was used to transduce various human cancer cell lines. RESULTS: Of eight randomly chosen G418-resistant clones generated by transfection, only two clone produced the vector at up to >10 (6) cfu/ml, while one of five clones generated by infection yielded higher-titer virus in the absence of helper virus, up to 1 X 10 (7) cfu/ml, than the transfected clones. Transduction with supernatant derived from a PA317 producer cell line has resulted in transduction levels from 1% to 15%, 5- to 60-fold lower than those analyzed in NIH3T3 cells. CONCLUSION: These findings suggest that new improved gene transfer method into human cancer cells using retroviruses is required for efficient in vivo cancer gene therapy.
Cell Line* ; Clone Cells ; Genes, Neoplasm ; Genetic Therapy ; Helper Viruses ; Humans* ; Lac Operon ; Product Packaging ; Retroviridae ; Transfection ; Zidovudine*

BACKGROUND: The basic treatment of malignant tumors is surgery, radiotherapy, chemotherapy. Even though, the object of these treatments is to kill cancer cells, they have limitations. So, in future studies of treatment of cancer, we should look into increasing human immune response using gene therapy in order to induce damage to tumor cells. OBJECTIVE: The cell growth inhibitory effect of cervical cancer cells was investigated by direct transfection using liposome(pRcCMVp53/lipofectin). and by indirect transfection using Adenovirus(AdCMVp53). METHODS: The cervical cancer cell lines we used in this study were HPV16 positive, having inhibitory gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3, LacZ gene was used as the marker gene for the transfection efficacy. Direct transfection was done by using lipofectin (pRcCMVp53/lipofectin) and indirect transfection was done by using virus, AdCMVp53. The effect of tumor cell growth inhibition was measured by cell counting assay. RESULT: Inhibition of growth of cervical cancer cells in cell counts of direct transfection was CaSki(88.5%), SiHa(59.1%), HeLa(86.0%), HeLaS3(78.0%), C33A(91.3%) and HT3(74.0%). Inhibition of growth of cervical cancer cells in cell counts of indirect transfection was CaSki(97.4%), SiHa(91.6%), HeLa(95.8%), HeLaS3(99.7%), C33A(97.3%) and HT3(87.4%). CONCLUSION: The inhibition of cell growth of cervical cancer cells by direct and indirect transfection was significantly reduced, and showed little differences depending on the type of cells. These results will have a great meaning in treating cervical cancer patients using gene therapy by direct or indirect transfection
Adenoviridae* ; Cell Count ; Cell Line ; Drug Therapy ; Genes, p53 ; Genetic Therapy* ; Humans ; Lac Operon ; Plasmids* ; Radiotherapy ; Transfection ; Uterine Cervical Neoplasms*

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5' flanking regions of human DSPP gene (- 4 000-+54, -2 500-+54, -1 447-+54 and -1 027-+54) were analyzed, the results showed that the highest promoter activity lied in the -2 500-+54 region. The promoter activity is reduced when the 5' flanking region was extended from -2 500 to -4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5' flanking regions were shorted from -2 500 to -1 447 bp and from -1 447 to -1 027 bp, indicating that potential suppresser elements are lied in the region between -4 000 and -2 500 bp and potential activator elements are lied in the region between -2 500 and -1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the -2 500-+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.
Cell Differentiation ; Extracellular Matrix Proteins ; genetics ; Genes, Reporter ; Humans ; Lac Operon ; Odontoblasts ; cytology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sialoglycoproteins ; genetics

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

OBJECTIVES: Brain transplantation has emerged as an effective treatment for patients suffering from neurodegenerative diseases, including Parkinson's disease, Huntingtons disease and Stroke. We evaluated that cytokine inducted human mesenchymal stem cells (Ci-hMSCs) transplanted in brain differentiated into neural cells and improved neurological functions after stroke in rats. MATERIALS AND METHODS: In the adult female Sprague-Dawley rats, ischemic lesion was induced by transient MCA occlusion lasted for 2 hours. One day later, Ci-hMSCs carrying LacZ gene were implanted via tail vein. The animals were assessed for sensorymotor function and sacrificed for Immunohistochemical staining at 7, 14, 28, 56 days after transplantation. RESULTS: A large number of X-gal positive hMSCs were observed in the ischemic core and ischemic boundary zone. Some hMSCs were reactive for the astrocytic marker - glial fibrillary acidic protein (GFAP) and neuronal marker - neuronal nuclear antigen (NeuN). The ischemic rats that were transplanted with Ci-hMSCs exhibited better functional improvement than control groups and the rats with hMSCs, which was statistically significant. CONCLUSION: The neuronal differentiation of CihMSCs suggested that transplantation of the Ci-hMSCs may provide the possibility of the clinical implication for cerebral stroke.
Adult ; Animals ; Brain ; Female ; Glial Fibrillary Acidic Protein ; Humans* ; Lac Operon ; Mesenchymal Stromal Cells* ; Models, Animal* ; Neurodegenerative Diseases ; Neurons ; Parkinson Disease ; Rats* ; Rats, Sprague-Dawley ; Stroke ; Veins

OBJECTIVETo explore a way of the gene therapy for acute spinal cord injury (ASCI) by vivo transfection of exogenous gene into spinal cord tissue.

METHODSTwenty-four rats of SD were divided into experiment group and control group (each group had 12 rats). After anaesthesia by abdominal cavity, lamina of thoracic vertebra of all rats were cut-open in prone position. Complex of plasmid and report gene-Lac Z, and plasmid without report gene-Lac Z were respectively injected into cavum subdural of SD rats of experiment group and control group by cation liposome (DOTAP) encapsulation. The rats were killed at the 2nd week after operation, spinal cord tissue of injected segments were detected by reverse transcription-polymerase chain raction (RT-PCR) and immunohistochemistry.

RESULTSIn experiment group, positive staining of beta-galactosidase can be clearly observed in neuron and glia cell of rat's spinal cord by immunohistochemistry detection. Lac Z mRNA in same area was also detected by RT-PCR. But, in control group, no above-mentioned positive results were found.

CONCLUSIONEffective transfection of exogenous gene in vivo into spinal cord is a new hot spot for treatment of SCI. Thus certain nerve growth factor imput partly area of spinal cord injury can promote central nerve regrowth and avoid early secondary injury.

Acute Disease ; Animals ; Genetic Therapy ; Lac Operon ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; therapy ; Transfection ; beta-Galactosidase ; analysis

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Escherichia coli ; genetics ; Gene Knock-In Techniques ; methods ; Gene Knockout Techniques ; methods ; Humans ; Lac Operon ; genetics ; Plasmids ; genetics ; Recombination, Genetic