1.Changes of Transfection Efficiencies of Plasmid LacZ Genes in Human Gynecologic Cancer Cell Lines.
Byung Hoon KIM ; Su Mi BAE ; You Jin HAN ; Hyeon Seung LEE ; Yoon Sung JO ; Keun Ho LEE ; Soo Young HUR ; Jae Hoon KIM ; Young Wook KIM ; Duck Yong RO ; Joon Mo LEE ; Sung Eun NAMKOONG ; Dong Jae KIM ; Chong Kook KIM ; Woong Shick AHN
Korean Journal of Obstetrics and Gynecology 2004;47(5):870-879
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*
2.Promoter recognition and beta-galactosidase reporter gene expression in Rhodococcus.
Changchun LIU ; Huimin YU ; M YUCHAO ; Wenyu PAN ; Hui LUO ; Zhongyao SHEN
Chinese Journal of Biotechnology 2009;25(9):1360-1365
The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.
Escherichia coli
;
genetics
;
Gene Expression Regulation, Bacterial
;
Genes, Reporter
;
genetics
;
Lac Operon
;
genetics
;
Promoter Regions, Genetic
;
genetics
;
Rhodococcus
;
enzymology
;
genetics
;
beta-Galactosidase
;
genetics
3.Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination.
Shanhu LI ; Qingguo SHI ; Cuifen HUANG ; Jianguang ZHOU
Chinese Journal of Biotechnology 2008;24(4):576-580
To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda
;
genetics
;
Chromosomes, Bacterial
;
genetics
;
Cloning, Molecular
;
Escherichia coli
;
genetics
;
metabolism
;
Gene Knock-In Techniques
;
methods
;
Genes, Reporter
;
genetics
;
Lac Operon
;
genetics
;
Luciferases
;
genetics
;
Recombination, Genetic
;
genetics
5.New Approach to the Analysis of Palindromic Structure in Genome Sequences.
Seok Won KIM ; Yong Seok LEE ; Sang Haeng CHOI ; Sung Hwa CHAE ; Dae Won KIM ; Hong Seog PARK
Genomics & Informatics 2006;4(4):167-169
PABAP (Palindrome Analysis by BLAST Program) is an analysis system that identifies palindromic sequences from a large genome sequence up to several megabases long. It uses NCBI BLAST as a searching engine, and data processing such as alignment filtration and detection of inverted repeats which satisfy user- defined parameters is performed by manipulating data after populating into a MySQL database. PABAP outperforms publicly available palindrome search program in that it can detect large palindrome with internal spacer at a faster speed from bacterial genomes. It is a standalone application and is freely available for noncommercial users. AVAILABILITY: This application was implemented with free software (Perl, Apache, MySQL, and NCBI BLAST) and is freely available to noncommercial users upon request. Analysis of user data can be carried out directly at http://chimp.kribb.re.kr/~javamint/palindrome.
APACHE
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Filtration
;
Genome*
;
Genome, Bacterial
6.Lactoferrin as a gene delivery vehicle to hepatocytes.
Sang Taek OH ; Jeong Keun RIH ; Heung Sun KWON ; Deog Su HWANG ; Sun Young KIM ; Jeong Bin YIM
Experimental & Molecular Medicine 1997;29(2):111-116
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
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Cats
;
Chromatography
;
Dialysis
;
Fibroblasts
;
Filtration
;
Genes, Reporter
;
Genes, vif*
;
Hepatocytes*
;
Humans
;
Lac Operon
;
Lactoferrin*
;
Liver
;
Mice
;
Polylysine
;
Transfection
7.Heterologous Regulation of BCG hsp65 Promoter by M. leprae 18 kDa Transcription Repression Responsive Element.
Genomics & Informatics 2003;1(2):113-118
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*
8.Expression of the reporter LacZ driven by human dentin sialophosphoprotein promoter in human dental mesenchymal cells.
Meizhen LIN ; Meiqin JIANG ; Shuiqin LI ; Yan LIN ; Yide HUANG
Chinese Journal of Biotechnology 2016;32(8):1133-1144
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
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Extracellular Matrix Proteins
;
genetics
;
Genes, Reporter
;
Humans
;
Lac Operon
;
Odontoblasts
;
cytology
;
Phosphoproteins
;
genetics
;
Promoter Regions, Genetic
;
Sialoglycoproteins
;
genetics
9.Comparison of Efficiency of Infection of Human Cancer Cell Lines Via Retroviral Vector System.
Yeon Soo KIM ; Hee Kyung LIM ; Joo Hang KIM ; Jin Sik MIN
Journal of the Korean Cancer Association 1997;29(1):1-10
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*
10.Development of Gene Therapy Strategy Using Plasmid and Adenovirus in Cervical Cancer Treatment.
Jun Mo LEE ; Seung Jo KIM ; Sung Eun NAMKOONG ; Sung Dae CHO ; Seong Jin HWANG ; Hyun Ra PARK ; You Jin HAN ; Sang Tae KIM ; Hun Young LEE ; Dong Jae KIM ; Yong Serk PARK ; Chong Kook KIM ; Yu Kyoung OH ; Soon Hee PARK ; Woong Shick AHN
Korean Journal of Obstetrics and Gynecology 1999;42(9):2019-2027
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*