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

OBJECTIVETo screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex.

METHODSSixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing.

RESULTSAmong 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR.

CONCLUSIONSNATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.

Animals ; Cell Line ; Cerebral Cortex ; Mice ; Molecular Sequence Data ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics

Animals ; Humans ; RNA Interference ; RNA, Antisense ; genetics ; RNA, Small Interfering ; genetics ; SARS Virus ; genetics ; physiology ; Severe Acute Respiratory Syndrome ; virology

Child ; DNA, Antisense ; genetics ; DNA, Viral ; genetics ; Genetic Therapy ; methods ; Humans ; RNA, Antisense ; genetics ; Respiratory Syncytial Virus Infections ; therapy ; Respiratory Syncytial Virus, Human ; genetics ; pathogenicity

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

OBJECTIVETelomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse-transcript protein subunit (hTERT) is the key element and rate-limiting factor of telomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro.

METHODSThe sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions, then the HepG2-s and HepG2-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT.

RESULTSAntisense hTERT significantly down-regulated the endogenous hTERT mRNA expression (15.35 +/- 1.72/HepG2-as, 43.8 +/- 2.89/HepG2-s, 45.2 +/- 3.46/HepG2) (n = 10, t = 7.61, P < 0.01) and telomerase activity in HepG2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage-independent growth in HepG2-as were significantly suppressed (50.6 +/- 4.8/HepG2-as, 113.52 +/- 8.15/HepG2-s, 119.12 +/- 10.82/HepG2) (n = 10, t = 4.54, P < 0.01 and n = 10, t = 3.96, P < 0.01), compared to HepG2 and HepG2-s. However there was a significant increase in apoptosis percentage of HepG2-as by flow cytometry (n = 10, t = 9.24, P < 0.01 and n = 10, t = 8.37, P < 0.01), compared to control group.

CONCLUSIONSAntisense hTERT could significantly suppress the hepatoblastoma cell growth and reverse its malignant phenotypes in vitro and cause the increase in apoptosis percentage of HepG2, thus it might be applied in malignant tumor gene therapy through the telomerase-targeted molecular mechanism.

Cell Division ; genetics ; Cell Line, Tumor ; DNA-Binding Proteins ; Hepatoblastoma ; genetics ; pathology ; Humans ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics

With the development of genome-wide sequencing technology, 195 types of functional long non-coding RNAs (lncRNAs) have so far been found, and their cellular roles are gradually being revealed. Now lncRNAs have become a hotspot in the life science. These small molecules exist in almost all higher eukaryotes, and have very important regulatory roles in these organisms. This review briefly summarizes recent progress in researches on antisense non-coding RNA in the INK4 locus.
Animals ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; Genetic Loci ; Humans ; RNA, Antisense ; physiology ; RNA, Long Noncoding ; physiology

OBJECTIVETo explore the effects of survivin antisense RNA and HSP70 double gene transfection on breast cancer cell line MCF-7.

METHODSMCF-7 cells was transfected with the double-gene vector pIRES2-EGFP-survivin antisense RNA/HSP70 via liposome. After a 72-h transfection, the cells were collected for observation under inverted fluorescent microscope. The changes of survivin mRNA and HSP70 protein expressions in the cells were detected with real-time PCR and Western-blot before and after the cell transfection, and the apoptotic rate of the transfected MCF-7 cells was detected by flow cytometry analysis with Annexin-V-cy5/7AAD double staining.

RESULTSGreen fluorescence was detected in MCF-7 cells transfected with the double-gene expression vector and the empty vector under inverted fluorescent microscope. The expression level of survivin mRNA in the cells was reduced effectively after the transfection with the double-gene expression vector, which also induced obvious cell apoptosis and enhanced the expression level of HSP70 protein as compared with those in MCF-7 cells transfected with the empty vector and the untransfected MCF-7 cells.

CONCLUSIONSurvivin antisense RNA can interfere with the expression of endogenous survivin and induce apoptosis of MCF-7 cells. HSP70 can increase the expression of HSP70 protein in MCF-7 cells.

Apoptosis ; drug effects ; Female ; HSP70 Heat-Shock Proteins ; genetics ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; pharmacology ; MCF-7 Cells ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Transfection

OBJECTIVESTo investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells.

METHODSThe antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods.

RESULTSThe growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited.

CONCLUSIONThe expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.

Cell Division ; drug effects ; Humans ; K562 Cells ; Plasmids ; genetics ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Telomerase ; drug effects ; genetics ; metabolism ; Transfection