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

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

Filamentous fungi are important industrial microorganisms. The focus on its metabolic engineering is to optimize the metabolic pathway with gene expression regulation technology to meet with the industrial production needs. Antisense RNA technology due to its simplicity compared with the gene knock-out technology has great perspectives in filamentous fungal metabolic control. It is an efficient method for regulating gene expression and a key tool for metabolic engineering. In this article, we addressed the mechanism of antisense RNA technology and its applications in filamentous fungal metabolic engineering. Additionally, future perspectives were discussed.
Fungi ; genetics ; Gene Expression Regulation, Fungal ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; Metabolism ; RNA, Antisense ; genetics ; 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

OBJECTIVETo explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.

METHODSAccording to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.

RESULTSBoth the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.

CONCLUSIONSThe cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

DNA, Antisense ; genetics ; Genome, Viral ; Hepatitis B virus ; genetics ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; RNA, Catalytic ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Transcription, Genetic

BACKGROUNDTo construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection.

METHODSThe DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell.

RESULTSThe psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA.

CONCLUSIONSThe gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.

3T3 Cells ; Animals ; Eukaryotic Cells ; metabolism ; Gene Expression ; Genetic Vectors ; Mice ; Plasmids ; genetics ; RNA, Antisense ; genetics ; Receptors, CCR5 ; genetics ; Transfection

OBJECTIVESTo study the inhibitory effects of mouse telomerase RNA (mTR) antisense oligodeoxynucleotide(ASODN) on telomerase activity in rat spermatogonia.

METHODS9-mer phosphorothioate mTR-ASODN was encapsulated by Lipofect AMINE 2000 (LF 2000) and transfected to type A spermatogonia in Snrague Dawley (SD) rat. Telomerase activity was detected by aid of TRAP-SYBR-Green staining and Bioluminescence technique in type A spermatogonia treated or untreated with ASODN.

RESULTSmTR-ASODN conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia(P < 0.01). mTR mRNA level also decreased while the spermatogonia were treated with ASODN for 24 h. No change of telomerase activity and apoptosis were observed when SODN, RODN or single LF 2000 was used.

CONCLUSIONSAntisense oligodeoxynucleotide of mTR conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia. mTR-ASODN might inhibit telomerase activity of spermatogonia at transcription level.

Animals ; Male ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; enzymology ; ultrastructure ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVESElevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.

METHODSAntisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.

RESULTSThe results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).

CONCLUSIONRNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.

Animals ; Cells, Cultured ; Dependovirus ; genetics ; Genetic Vectors ; Hepatic Stellate Cells ; metabolism ; RNA, Antisense ; genetics ; RNA, Small Interfering ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON and tTG-ASDON2 were significantly decreased as compared with that of the controls (P < 0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.
Cells, Cultured ; Glaucoma, Open-Angle/metabolism ; Oligonucleotides, Antisense/*pharmacology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*metabolism ; Transglutaminases/*biosynthesis ; Transglutaminases/genetics ; Transglutaminases/*pharmacology

OBJECTIVETo observe the role of corticotropin releasing factor receptor 2 antisense oligodeoxynucleotide (CRFR2ASO) of hypothalamus in hypermetabolism in rats with severe burn.

METHODSStainless-steel cannula were implanted into the 3rd ventricle. According to different medicine delivered into the 3rd ventricle, 30 SD rats with 30% TBSA full-thickness burn were divided randomly into burn control group (BC, with injection of 3 microL saline), CRFR1ODN group (with injection of CRFR1ODN 10 microg), CRFR1ASO group (with injection of CRFR1ASO 10 microg), CRFR2ODN group(with injection of CRFR2ODN 10 microg), CRFR2ASO group (with injection of CRFR2ASO 10 microg), with 6 rats in each group. Another 6 rats served as normal control (NC) and they received isotonic saline 3 microL instead. Different medicines were respectively delivered into respective group on 5, 6 post injury day (PID), then 3 microL gentian violet was introduced on 7 PID. Resting energy expenditure (REE) value and the expression level of hypothalamus CRFR2mRNA and CRFR2 protein were determined.

RESULTSREE value in BC, CRFR1ODN, CRFR1ASO, CRFR2ODN, CRFR2ASO groups was 11 840 +/- 987, 11 133 +/- 1100, 10 733 +/- 1338, 11 123 +/- 1321, 7563 +/- 890 kJx(m2)(-1)xd(-1), respectively, which were obviously higher than that in BC group [6641 +/- 526 kJx(m2)(-1)xd(-1), P < 0.05]. REE value in CRFR2ASO group was obviously lower than that in CRFR2ODN group (P < 0.01). The expression level of hypothalamus CRFR2 mRNA and its protein in BC group were increased after burn, which were obviously lower in CRFR2ASO group than NC group (P < 0.01).

CONCLUSIONCentral application of CRFR2ASO can downregulate the expression level of hypothalamus CRFR2 mRNA and its protein, and reduce hypermetabolism. Hypothalamus CRFR2 may mediate hypermetabolism in burn rats.

Animals ; Burns ; metabolism ; Hypothalamus ; metabolism ; Male ; Oligodeoxyribonucleotides, Antisense ; metabolism ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Corticotropin-Releasing Hormone ; genetics ; metabolism