Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.
Amino Acid Sequence ; Base Sequence ; Drug Resistance ; genetics ; Genetic Markers ; Genetic Vectors ; Kanamycin Kinase ; genetics ; Molecular Sequence Data ; Mutation

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.
Adenosine ; Amikacin ; Amphotericin B ; Anti-Bacterial Agents ; Diffusion ; Gentamicins ; Kanamycin ; Kanamycin Kinase ; Methicillin ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Multiplex Polymerase Chain Reaction ; Netilmicin ; Plasmids ; Sprains and Strains ; Staphylococcus aureus ; Tobramycin

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.
Adenosine ; Amikacin ; Amphotericin B ; Anti-Bacterial Agents ; Diffusion ; Gentamicins ; Kanamycin ; Kanamycin Kinase ; Methicillin ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Multiplex Polymerase Chain Reaction ; Netilmicin ; Plasmids ; Sprains and Strains ; Staphylococcus aureus ; Tobramycin

BACKGROUND: Human cells are almost never spontaneously immortalized in vitro. We tried to immortalize human fetal hepatocytes (h-FH) and evaluate the differentiational status and its change. METHODS: Hepatocytes were isolated from a liver fragment of 20 week old fetus and infected with amphotropic recombinant retrovirus containing a temperature- sensitive mutant of SV40 large T antigen and neomycin phosphotransferase gene. G418 resistant colonies were cloned and expanded. The cells which were able to divide more than 30 times were used to analyze various functions. RESULTS: The immortalization rate was 3.3 x 10-8 and two cell lines (C11, D21) were established. C11-60, C11-80, D21-30 and D21-60 (suffix number means the cell division counts) were evaluated. D21-30 was thougt to be imcompletely immortalized because a considerable portion of cells died during culture. The morphology was similar to that of epithelial cells except for D21-30 which looked like fibroblast. The cells grew rapidly at 33oC but stopped growing at 39oC. T antigen and p53 was expressed at 33oC but disappeared at 39oC, which suggest that T antigen binds to p53. Chromosomal changes were so marked that it was impossible to discriminate exact number. Albumin was secreted as about 1/10 as that of h-FH, but alpha-fetoprotein secretion stopped after immortalization. Telomerase was activated in both cell lines except for the incompletely immortalized cells D21-30. Telomere was elongated in competely immortalized cell lines, but it was rather shortened in D21-30 compared to that of h-FH. Macroscopic colonies did not develop in soft agar assay. CONCLUSIONS: We successfully immortalized human fetal hepatocytes. Although the cells are not likely to have oncogenicity, the functions are not so good, possibly due to marked chromosomal changes which are thought to occur before telomerase is activated during immortalization step.
Agar ; alpha-Fetoproteins ; Antigens, Viral, Tumor* ; Cell Division ; Cell Line ; Clone Cells ; Epithelial Cells ; Fetus ; Fibroblasts ; Hepatocytes* ; Humans* ; Kanamycin Kinase ; Liver ; Retroviridae* ; Telomerase ; Telomere

BACKGROUND: The aim of this study was to evaluate the prevalence of IgE sensitization and allergic risk of genetically modified (GM) potato compared with wild one in adult patients with various allergic diseases. METHODS: One thousand eight hundred eighty eight allergy patients visited Ajou University hospital and 38 healthy controls were enrolled. Skin prick tests were performed with wild and GM extracts. Phosphinothricin N-acetyltransferase (PAT) and neomycin phosphotransferase (NPT) gene was inserted in GM potato. Serum specific IgE level to the two potato extracts was measured by ELISA and their binding specificities were confirmed by ELISA inhibition test. IgE binding components in both wild and GM potato extracts were identified by SDS-PAGE and IgE-immunoblot. RESULTS: One hundred eight patients (5.7%) showed positive responses (A/H >or= 2+) on skin prick test to both wild and GM potatoes. Serum specific IgE was detectable in 50~88% among the positive reactors on skin prick test. ELISA inhibition tests showed similar inhibition pattern between wild and GM. Fourteen IgE binding components within wild potato and nine IgE binding components within GM potato with similar binding patterns, of which three major allergens in wild (26, 34, 45 kDa) and one (45 kDa) in GM one were noted.. CONCLUSIONS: The sensitization rates to wild and GM potato extracts were 5.7% respectively, in adult allergy patients and one common major allergen (45 kDa) was identified. It is speculated that genetic manipulation of the potato did not increase allergenic risk.
Adult* ; Allergens ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Food, Genetically Modified ; Humans ; Hypersensitivity* ; Immunization ; Immunoglobulin E* ; Kanamycin Kinase ; Prevalence ; Skin ; Solanum tuberosum*

PURPOSE: Retinoids have been shown to be effective in suppressing tumor development when chemical carcinogens such as N-nitroso-N-methylurea (NMU) and N- nitroso-N-ethylurea (NEU) were used to induce mammary tumors in a variety of animal models. However, the molecular mechanisms associated with the retinoid- mediated chemopreventive process, as linked to transcription factor NF-kappa B activation, for chemoprevention have not been elucidated. The purpose of this study was to determine the implications of NF-kappa B activation on the chemopreventive role of retinoids and their effect on cellular NF-kappa B activity that's induced by known alkylating chemical carcinogens such as NMU and NEU in human transfectant squamous cell carcinoma (SCC-13) cells. MATERIALS AND METHODS: The activity of NF-kappa B, as regulated by chemical carcinogens and retinoids, was determined in cultured human SCC-13 keratinocytes that were transfected with the pNF-kappa B-SEAP-NPT plasmid; this permitted the expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappa B activity, and the plasmid contained the neomycin phosphotransferase (NPT) gene, which confers resistance to geneticin. The reporter enzyme activity was measured using a fluorescence detection assay method. RESULTS: All-trans retinoic acid and 13-cis retinoic acid induced a reduction of NF-kappa B activity up to 64% and 65%, respectively, compared to the control. For the treatment of the human transfectant cells with chemical carcinogens, all-trans retinoic acid (5 mM) and 13-cis retinoic acid (5 mM) downregulated the cellular NF-kappa B activation up to 83% and 85% compared to the NF-kappa B activity that was upregulated by NMU (5 micro M) and NEU (5 micro M), respectively. CONCLUSION: These results suggest that the chemopreventive effect of retinoids may be mediated by the down- regulated activation of NF-kappa B and that retinoids are implicated in the activation of NF-kappa B in human skin cells.
Alkaline Phosphatase ; Carcinogens ; Carcinoma, Squamous Cell ; Chemoprevention ; Fluorescence ; Genes, Reporter ; Humans* ; Kanamycin Kinase ; Keratinocytes* ; Models, Animal ; NF-kappa B* ; Plasmids ; Retinoids* ; Skin ; Transcription Factors ; Tretinoin

OBJECTIVETo determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens.

METHODSThe high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates.

RESULTSThe ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant.

CONCLUSIONHLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus ; drug effects ; genetics ; Enterococcus faecalis ; drug effects ; genetics ; Enterococcus faecium ; drug effects ; genetics ; Gentamicins ; pharmacology ; Humans ; Kanamycin Kinase ; genetics ; Microbial Sensitivity Tests