With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.
Bacteria ; genetics ; metabolism ; Genes, Bacterial ; Genetic Engineering ; Metabolic Networks and Pathways ; genetics ; Oligonucleotides, Antisense ; genetics ; RNA, Antisense ; genetics ; RNA, Catalytic ; genetics

The study was supposed to investigate the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide (ASPSODN) targeting hTERT mRNA on gene of interest in K562 cells and influence of ASPSODN on telomerase activity and apoptosis of K562 cells. Human leukemia cell line K562 was transfected by liposome with ASPSODN and SPSODN (sense phosphorothioate oligodeoxynucleotide) at different concentrations (0.2, 0.6, 1.0 micromol/L). At the same time, blank control, liposome control and SPSODN groups were designed for comparison. The transfected cells were collected and detected at 24 and 48 hours; the expression of target gene hTERT mRNA and telomerase activity were detected by RT-PCR and TRAP-ELISA respectively, and cell apoptosis was assayed by flow cytometry. The results showed that after K562 cells were transfected for 24 hours, the expression of hTERT mRNA had no difference between liposome control (0.80+/-0.24), 0.2 micromol/L ASPSODN (0.69+/-0.12), 0.2 micromol/L SPSODN (0.72+/-0.25) and blank control (0.85+/-0.28), but the expression of hTERT mRNA in 0.6 micromol/L ASPSODN group (0.42+/-0.16) remarkably decreased as compared with liposome control group, 0.6 micromol/L SPSODN (0.69 +/- 0.26) had no obvious effect on the expression of hTERT mRNA, the expression of hTERT mRNA in 1.0 micromol/L ASPSODN and SPSODN groups both decreased; mortality of K562 cells transfected by liposome with 1.0 micromol/L ASPSODN and SPSODN remarkably increased. After 24 hours, telomerase relative activity of K562 cells showed no significant difference between blank control (88.9%) and liposome control (77.7%). The telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 60.6%, 52%, 58.2% respectively. There was significant difference as compared with blank control; 0.6 micromol/L ASPSODN showed significant difference (p=0.037), as compared with liposome control group. The telomerase relative activities in K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN were 76.1%, 72.2%, 65.7% respectively, but the telomerase relative activities of K562 cells in 0.2, 0.6 micromol/L SPSODN groups was not inhibited obviously. When K562 cells were treated for 48 hours, telomerase relative activity of K562 cells in each ASPSODN groups restored. It showed that telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 84.1%, 82.3%, 79.6% respectively, while telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN for 48 hours were 74.8%, 74.5%, 67.9% respectively. Telomerase activity of K562 cells could not be inhibited by 0.2 and 0.6 micromol/L SPSODN. After culturing for 48 hours, the cell apoptosis rates of K562 in 0.6 micromol/L ASPSODN, 0.6 micromol/L SPSODN, liposome control and blank control groups were (4.82+/-0.39)%, (1.83+/-0.34)%, 1.84+/-1.04)%, (1.07+/-0.74)% respectively. There was difference between ASPSODN and SPSODN groups (p<0.05), but the significant difference was found in ASPSODN group as compared with liposome control and blank control (p<0.01). It is concluded that the ASPSODN targeting hTERT can specifically inhibit the expression of hTERT mRNA in K562 cells and significantly suppress the telomerase activity of K562 cells at 0.6 micromol/L, which inhibitory time is short. The ASPSODN at high concentration (1.0 micromol/L) shows definite cytotoxicity. 0.6 micromol/L of ASPSODN significantly induces cell apoptosis, while no such effect was seen in SPSODN group.
Apoptosis ; drug effects ; Humans ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; Telomerase ; genetics ; metabolism

In order to study the effects of phosphorothioated antisense oligodeoxynucleotides (ASODN) on the expression of VEGF in human lymphoma cell line Namalwa cells, human lymphoma cell line Namalwa cells were incubated with VEGF ASODN (the final concentrations of VEGF ASODN were 5, 10, 20 micromol/L respectively), or scrambled sequence for 24 or 48 hours. The expressions of VEGF mRNA and VEGF protein were detected by reverse transcriptase-polymerase chain reaction and streptavidin peroxidase (SP) immunohistochemistry respectively. The results showed that the expression levels of VEGF mRNA in Namalwa cells treated with three concentration levels (5, 10, 20 micromol/L of ASODN) were 1.38, 0.96 and 0.57 respectively. Those in PBS-treated cells and scrambled sequence treated cells were 1.79 and 1.84. When treated with 20 micromol/L VEGF ASODN for 48 hours, VEGF protein of Namalwa cells decreased greatly. Meanwhile, there was no obvious change in the scrambled sequence treated group. It is concluded that VEGF ASODN can suppress the VEGF expression in Namalwa cells in vitro.
Cell Line, Tumor ; Humans ; Lymphoma ; metabolism ; pathology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVE: To determine the effect of EphA2 protein on the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) proteins in HCT116 cells. METHODS: High expression of EphA2 protein in HCT116 cells was confirmed by Western blot. HCT116 cells were transfected with EphA2 antisense oligonucleotide. The expression of the transfection efficiency was analyzed by Western blot. VEGF proteins in the cell supernatants were detected by enzyme linked immunosorbent assay(ELISA), and the expressions of MMP9 in cell supernatants were examined by gelatin zymography. RESULTS: EphA2 antisense oligonucleotide suppressed the expression of VEGF and MMP9 proteins in HCT116 cells. CONCLUSION EphA2 could decrease the invasion and metastasis of HCT116 cells by suppressing the expression of VEGF and MMP9.
HCT116 Cells ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Oligonucleotides, Antisense ; Receptor, EphA2 ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

To study the pharmacokinetics of cantide, an antisense oligonucleotide, and its metabolites after iv gtt administration in rhesus monkeys, a dual solid phase extraction pretreatment method coupling with non-gel sieving capillary electrophoresis analysis method was used for determination of cantide and its metabolites in plasma and their pharmacokinetic parameters were calculated. The pharmacokinetic behavior of cantide and its metabolites (M1 and M2) after iv gtt administration (8, 16 and 24 mg kg(-1)) in rhesus monkeys were investigated. After iv gtt administration of cantide to rhesus monkeys, cantide in plasma was eliminated rapidly and the terminal elimination half-life (t1/2) was 57.91-77.97 min, the correlation coefficients (r) to the dose of Cmax AUC(o-inf) and AUC(0-t) of the prototype was 0.9918, 0.9568 and 0.9773, respectively. The metabolites of cantide reached the Cmax following cantide immediately and the Cmax of metabolites were lower than that of the prototype. The CL(S) of cantide and its metabolites (M1 and M2) were 1.60-2.19, 5.92-8.58 and 6.07-8.78 mL min(-1) kg(-1), respectively. So, it is concluded that the Cmax of cantide and its metabolites increased with the dose, which is the same as their AUC(0-inf) and AUC(0-t). The CL(S) of metabolites were higher than that of the prototype. The MRT and t1/2 of metabolites in the high dose group increased obviously.
Animals ; Area Under Curve ; Electrophoresis, Capillary ; methods ; Female ; Half-Life ; Infusions, Intravenous ; Macaca mulatta ; Male ; Oligonucleotides, Antisense ; blood ; metabolism ; pharmacokinetics ; Phosphorothioate Oligonucleotides ; blood ; metabolism ; pharmacokinetics ; Solid Phase Extraction

The objective of study was to investigate the effect of liposomal transfection of antisense oligodeoxynucleotide (ASON) on alpha-globin gene expression and proliferation of K562 cells, to explore the new way of gene therapy in beta-thalassemia. Targeted ASON of alpha-globin was designed and synthesized, and compared with positive control [sense oligodeoxynucleotide (SON) group] and blank control. By liposomal transfection, ASON, SON was co-cultured with K562. The efficiency of transfection was assayed by fluorescence microscopy and flow cytometry (FCM), the alpha-globin gene expression of K562 was measured by real-time PCR, and the proliferation of K562 was determined by Cell Count Kit-8 assay. The results indicated that the highest efficiency was at 24 hours after liposomal transfection, the gene expression level of alpha-globin in ASON group was significantly lower than that in SON group and blank control (p < 0.01). The proliferation of K562 cells was obviously inhibited, meanwhile the above effect showed the dose-dependent manner. It is concluded that the liposomal transfection of ASON inhibits the alpha-globin gene expression of K562 cells, which may be the new target for gene therapy in beta-thalassemia.
Cell Proliferation ; drug effects ; Gene Expression ; Humans ; K562 Cells ; Liposomes ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; Transfection ; alpha-Globins ; metabolism

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

Bcr-abl antisense oligodeoxynucleotides (AS-ODNs) have provided evidence of an antileukemia effect when tested in vitro against Philadelphia-positive cells. In order to investigate the efficacy of AS-ODNs as purging agents in chronic myeloid leukemia (CML) patients, K562 cells, a human CML cell line, were treated in vitro with various types of AS-ODNs and interferon-alpha. Cells were treated in vitro for 0 and 36 hr with 40 microgram/mL of AS-ODNs, respectively, and incubated at 37 degrees C for 36 hr. Cytotoxic effects were measured by counting the number of viable cells as well as by MTT test. Clonogenic activities were evaluated by methylcellulose culture for 2 weeks. The effects of purging agents on the rearrangement of bcrabl gene were evaluated by RT-PCR. AS-ODNs inhibited the proliferation of K562 cells with time in cell count assay and MTT test. AS-ODNs were superior to INF-alpha in inhibiting clonogenic activity (recovery rate; 26.3% vs 64.0%). After incubation with bcr-abl AS-ODNs primers and mRNA isolated from K562 cells, positive bands were abolished, especially of b3a2 type and phosphorothioate type. Our results suggest that AS-ODNs mediated purging may be one of the efficient methods and that autograft may be an alternative treatment for allograft in high-risk group patients of CML if they do not have a stem cell donor.
Bone Marrow Purging* ; Colony-Forming Units Assay ; Fusion Proteins, bcr-abl/genetics ; Fusion Proteins, bcr-abl/metabolism ; Hematopoietic Stem Cells/physiology* ; Human ; Leukemia, Myeloid, Chronic/therapy ; Neuroblastoma/therapy* ; Oligonucleotides, Antisense/metabolism* ; Oligonucleotides, Antisense/therapeutic use ; Transplantation, Autologous* ; Tumor Cells, Cultured

OBJECTIVETo explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid.

METHODSCTGF antisense oligonucleotides (ASODN) conjugated with isothiocyanate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblast (HKF) culturing media. The intracellular distribution of CTGF ASODN was observed with fluorescence microscopy in the fixed HKF. The proliferation of HKF was measured by MIT test. The apoptosis of HKF was measured with a flow cytometer. The collagen synthesis of HKF was measured by using H3-proline incorporation method.

RESULTSThe CTGF ASODN inhibited the proliferation and collagen synthesis of the HKF, compared with the control, but it increased the apoptosis after the transfection (P < 0.01).

CONCLUSIONCTGF ASODN may has anti-fibrotic effects on human keloid in vitro, and the CTGF may play an important role in promoting the fibrosis of human keloid.

Apoptosis ; Cell Differentiation ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; Fibroblasts ; cytology ; Humans ; Keloid ; metabolism ; pathology ; Oligonucleotides, Antisense ; genetics ; Transfection

Apoptosis ; drug effects ; Hep G2 Cells ; Hepatoblastoma ; metabolism ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-mdm2 ; genetics