Recombinant Plasmid Vector Research Articles

Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate, which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases. The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3′-OH group and terminates the process of DNA replication, hence leading to cell apoptosis. At present, HSVtk gene usually acts as suicide gene to kill tumor cells. The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the α-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro. Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed. HL-7702 liver cells, HUH-7 HCC, and HepG2 HCC were transfected with the recombinant plasmids. HSVtK gene expression was detected using Western blotting analysis. HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent. The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added. Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully. HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells. After G418 selection, a HepG2 cells line stably expressing HSVtk gene was acquired. With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P <0.05). Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Effect of transfection of microRNA-10b and antisense microRNA-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1

Objective To observe the effect of microRNA (miR)-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1.Methods The recombinant eukaryotic expression plasmid vector with miR-10b sequence [pPG-CMV-enhanced green fluorescent protein (EGFP)-miR-10b] or the antisense sequence was transfected into human pancreatic cancer cell line (AsPC-1) with lipofectamine,and the efficiency of the instant expression was confirmed by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and fluorescence microscope.The difference of the migration and invasion of the cells was evaluated by matrigel assay,and the expression of RhoC was evaluated by Western blotting.Results The expression of miR-10b in the sense group was increased by 10 folds,and in antisense group,the expression was decreased to 14％ compare to the control (P ＜ 0.05).The expression of RhoC protein in the sense group was (95.7 ± 8.5) unit,and that in the antisense group (28.5 ± 5.7) unit (P ＜0.05).As compared to the antisense group,the number of migrating and penerating cells was decreased from (85 ±7) and (35 ±9) to (32±5) and (8 ±3) in the sense group (P＜0.05) Conclusion miR10b can up-regulate the expression of RhoC,and promote the migration and invasion of the AsPC-1 cells. Key words: MicroRNA-10b; Pancreatic cancer ; Migration ; Invasion

Transformation Fava Beans by Agrobacterium using Chitinase, Glucanase and CryIA (b) genes

In this study two plasmid vectors that are appropriate for plant transformation were made by preparation of gene cassettes for β-1, 3-glucanase from barley, chitinase from bean and cryIA (b) from Bacillus thuringiensis (BT). Each of these genes were cloned under the control of the CaMV35S promoter and the Nos terminator in pBI121 binary vector. pBI-Chi and pBI-Glu and recombinant plasmid vectors were constructed via cloning of chitinase , β-1,3- glucanase and cryIA (b) genes, respectively, instead of the gus gene in T-DNA region of pBI121 vector. Construction of pBI-<em>ChiGlu </em>recombinant plasmid vector was performed by means of cloning both of the complete chitinase and glucanase gene cassettes in pBI121 vector, with the intention of production synergistic effects against fungal infection.pBI-ChiBt recombinant plasmid vector containing both of the complete chitinase and Bt gene cassettes was also constructed in order to contemporaneous plants resistance to pest pathogens and fungal in a single transformation event. pBI-ChiGlu and pBI-ChiBt that have been introduced into the <em>A. tumefaciens </em>strain LBA4404 that was subsequently used for transformation. Results indicate that embryogenic calli are well appropriate as objective material for <em>Agrobacterium tumefaciens</em>-mediated transformation in Faba bean.Seventeen well established shoots were transferred to new MLS medium including suitable antibiotics.Finally six independent transgenic plants were successfully rooted on kanamycin-containing selection media and then transferred to soil after 20 days .Four plants out of six putative transgenic plants displaied to contain the end part of the <em>chit</em> transgene and <em>nos </em>terminator.The corresponding piece, 700 bp of the <em>chit</em> gene, was amplified using specific primer.These putative transgenic plants were also be measured for the presence of the <em>bgn</em>13.1 and <em>cryIA (b) </em>genes by PCR using specific primers.Two pieces with expected sizes (1221 bp and 640 bp) for <em>bgn</em>13.1 and <em>cryIA (b) </em>genes subsequently was amplified from three the transgenic plants and other three plants had not these fragments.A group of virG primers was used to discover Agrobacterium pollution that might have threw off the selection. Expression of chit ,<em> </em>and <em>β-1,3-glucanase</em> genes at the transcriptional level in the transformed plants were proven by means of RT-PCR but expression of <em>cryIA(b)</em>was not proven<em>.</em>The expected dimension of the amplified cDNA pieces were identificated in the transformed plants,that confirming the permanent annexation and expression of T-DNA in the genome of kanamycin-screened plants.

Anti-cancer Effects of Glypican-3 on Huh-7 Hepatocellular Carcinoma Cells

Aim: Previous studies have suggested Glypican-3 (GPC3) could be a valuable diagnostic marker for hepatocellular carcinoma. This study examined the effects of overexpression of GPC3 on Huh-7 hepatoma cells. Methods: We constructed a recombinant plasmid vector pcDNA3.1 (+)-GPC3 for GPC3 overexpression studies in Huh-7 cells. RT-PCR and Western blotting were used to confirm GPC3 gene expression. Cell proliferation was evaluated by 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. Cell cycle progression and apoptosis were determined by flow cytometry using propidium iodide (PI) and Annexin V-FITC/PI staining, respectively. Cell migration and invasion were examined by Boyden Transewll and Matrigel assays. Results: GPC3 overexpression effectively inhibited proliferation, induced cell cycle arrest at S phase and increased apoptosis in Huh-7 cells. Furthermore, GPC3 overexpression significantly inhibited the migration and invasion ability of Huh-7 cells. Conclusion: Our results demonstrate that GPC3 could be a new therapeutic target for hepatocellular carcinoma

MicroRNA-143 Downregulates Interleukin-13 Receptor Alpha1 in Human Mast Cells

MicroRNA-143 (miR-143) was found to be downregulated in allergic rhinitis, and bioinformatics analysis predicted that IL-13Rα1 was a target gene of miR-143. To understand the molecular mechanisms of miR-143 involved in the pathogenesis of allergic inflammation, recombinant miR-143 plasmid vectors were constructed, and human mast cell-1(HMC-1) cells which play a central role in the allergic response were used for study. The plasmids were transfected into HMC-1 cells using a lentiviral vector. Expression of IL-13Rα1 mRNA was then detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western Blotting. The miR-143 lentiviral vector was successfully stably transfected in HMC-1 cells for target gene expression. Compared to the control, the target gene IL-13Rα1 was less expressed in HMC-1 transfected with miR-143 as determined by RT-PCR and Western Blotting (p < 0.05); this difference in expression was statistically significant and the inhibition efficiency was 71%. It indicates that miR-143 directly targets IL-13Rα1 and suppresses IL-13Rα1 expression in HMC-1 cells. Therefore, miR-143 may be associated with allergic reaction in human mast cells.

Suppression effect of expressing parvovirus H-1 nonstructural protein 1 gene on human gastric cancer cells

Objective To investigate the suppression effect of expressing parvovirus H-1 nonstructural protein 1 (NS1) gene on human gastric cancer cells and the possible mechanisms.Methods A recombinant enhanced green fluorescent protein (eGFP) labeled NS1 of parvovirus H-1 plasmid was constructed.Human gastric cancer cell line SGC7901 was transfected with recombinant plasmid (experiment group) or blank vector (negative control group) and blank control group was treated with equal amount of phosphate buffered saline (blank control group).After transfection,the distribution of fluorescent signal was observed under fluorescent microscope.The expression of NS1 at gene and protein level was measured.Cell growth curve of each group was drawn.The expression of cell senescence-associated β-galactosidase (SA-β-Gal) was tested.The changes of cell cycle were investigated by flowcytometry.Two groups' comparision was performed by t-test.Results After transfection,NS1 was expressed in SGC7901 cells at gene and protein level.Compared with negative control group,the fluorescent signal accumulated in cell nucleus in experiment group.The percentage of SA-β-Gal positive cell in experiment group ((30.5 ± 1.4) ％) was higher than that of negative control group ((4.4± 1.1) ％) and the difference was statistically significant (t =-12.931,P ＜ 0.01).The growth inhibition rate of SGC7901 cells from the first day to the fourth day was 45％,62％,73％ and 77％,respectively.The cell cycle of eGFP-NS1 expressed SGC7901 cells was arrested at G0/G1 phase.Conclusion Parvovirus H-1 NS1 play the role in cell nucleus of gastric cancer cell line SGC7901 and could make cell cycle arrested at G0/G1 phase,which effectively inhibited the proliferation SGC7901 cell. Key words: Stomach neoplasms; H-1 parvovirus; Viral nonstructural proteins; Genes, tumor suppressor; Cell aging; Cell cycle

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

ObjectiveTo study the role of bone marrow mesenchymal stem cells (BMSCs) in construction of vascularized engineered tissue. MethodshVEGF165 was amplified via RT-PCR before recombinant with pShuttle- green fluorescence protein;green fluorescent protein (GFP)-CMV. Then the recombinant shuttle plasmid was transfected into BMSCs with LipofectamineTM 2000 for packaging and amplifying. hVEGF165 mRNA expression in BMSCs cells was tested. Results: The sequence of hVEGF165 in pShuttle-GFP-hVEGF165 plasmid was confirmed by double-enzyme cleavage method and sequencing. hVEGF165 was highly expressed in BMSCs. ConclusionsThe GFP/hVEGF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells, which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

Suicide gene therapy for hepatocellular carcinoma cells by survivin promoter-driven expression of the herpes simplex virus thymidine kinase gene

The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant ‘bystander effect’ was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC.

SiRNA targeting survivin inhibits the growth and enhances the chemosensitivity of hepatocellular carcinoma cells

Survivin has become an attractive anticancer therapeutic target due to its important role in tumor cell viability and its selective expression in tumor cells. In the present study, we constructed a recombinant siRNA plasmid vector against survivin and stably transfected it into HepG2 and SMMC‑7721 hepatocellular carcinoma cells invitro. Semi-quantitative RT-PCR and western blotting were used to determine the expression of survivin mRNA and protein, respectively. Tumor cell proliferation was assessed by trypan blue exclusion. We evaluated the change in caspase-3 activity, and the rate of cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. Assessment of chemosensitivity was carried out by MTT assay. The results showed that transfection of survivin siRNA caused a significant inhibition of survivin mRNA and protein expression which was associated with cell growth inhibition, specific G0/G1 phase arrest, increased caspase-3 activity and enhanced chemosensitivity to cisplatin in both HCC cell lines. We suggest that suppression of survivin expression by RNAi attenuates the malignant phenotype of hepatocellular carcinoma cells, and may provide a novel approach for anticancer gene therapy.

Construction and use of a prokaryotic expression system for Helicobacter pylori AhpC

BackgroundHelicobacter pylori is an important pathogen responsible for human gastric problems like inflammation, ulcers and cancer. It is widely prevalent in developing countries with low socioeconomic status. Since the infection remains asymptomatic in most individuals, efforts for efficient diagnostic markers to identify high risk patients are warranted. In this study, we constructed an expression vector that overexpresses the H. pylori AhpC protein as a glutathione S-transferase fusion protein. We furthermore examined whether this recombinant fusion protein retained immunogenicity and thus would be useful as a diagnostic marker.FindingsThe full-length tsaA gene from H. pylori strain G27, which encodes AhpC, was cloned in plasmid vector pGEX-6P-2 to create the recombinant plasmid vector pGEX-tsaA. The nucleotide sequence of the clone showed 100% homology with corresponding published sequence of original gene. Over-expression of the target protein GST-AhpC was achieved in E. coli BL21 (DE3) cells by induction with isopropyl-beta-D-thiogalactoside (IPTG). GST-AhpC was extracted and identified using SDS-PAGE as a 52 kDa protein. Western blotting results using commercial antibodies against whole cell H. pylori showed that the fusion protein retained immunogenecity.ConclusionA recombinant prokaryotic expression system was successfully established with high expression efficiency for target fusion gene pGEX-tsaA. The expressed GST-AhpC protein showed immunoreactivity against commercial anti-H. pylori antibodies. This recombinant fusion protein can be developed as a diagnostic marker for screening patients with chronic H. pylori infections.

Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells

Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and cysteine protease inhibitor ( CPI ) gene from periodic Brugia malayi (Bm) and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 (+). pcDNA3.1 (+)/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE). Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass (Mr) of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell. Key words: Brugia malayi; Glyceraldehydes-3-phosphate dehydrogenases; Cysteine proteinase inhibitors; Recombination, genetic; HeLa cells

The construction of IGF-II P4 promoter-driven tk expression vector

To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro. Recombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis. Identification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively. The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.

Construction and identification of a yeast two-hybrid bait vector and its effect on the growth of yeast cells and the self-activating function of reporter genes for screening of HPV18 E6-interacting protein

By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A (600 nm) values of two yeast fluids were 0.98+/-0.03 and 0.99+/-0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-alpha-gal plates, while no colony could survive on SD/-His/-Trp/X-alpha-gal, SD/-Ade/-Trp/X-alpha-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

Enhanced expression of trophinin promotes invasive and metastatic potential of human gallbladder cancer cells

To study effects of trophinin on the metastatic potential of human gallbladder cancer cells and its potential mechanism. Expression of trophinin in the highly metastatic GBC-SDH(i) cells was investigated by real time RT-PCR and western blot. Recombinant expression plasmid vector of the human trophinin gene was constructed and transfected into GBC-SD cells. Effects of trophinin on the invasion of GBC-SD cells were investigated by adhesion assay and invasion assay in vitro. The siRNA was used to down-regulate the expression of trophinin. Some genes related to the invasion and metastasis of cancer were determined by real time RT-PCR and western blot. The pulmonary metastasis regulated by trophinin was determined in the nude mice. Overexpression of trophinin in GBC-SDH(i) cells was confirmed compared with its parental counterparts. Up-regulation of trophinin enhanced the in vitro invasion in the GBC-SD/TRO cells. The enhancement was associated with increasing integrin alpha3, MMP-7, MMP-9, and Ets-1 expression. The results were further demonstrated by RNA interference experiment in vitro. In in vivo study, we also demonstrated that trophinin-transfected gallbladder cancer cells had more pulmonary metastases than the vector-transfected one or its parental counterparts. Overexpression of trophinin leads to a more invasive phenotype and metastatic potential in human gallbladder cancer, at least in part, through regulating integrin alpha3, MMP-7, MMP-9, and Ets-1 expression.

Site-specific gene cassette insertion by combining artificial restriction DNA cutter and single-stranded DNA specific endonuclease

An artificial restriction DNA cutter (ARCUT), which was composed of Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acid, was used for construction of recombinant plasmid vector. The plasmid vector was cut by ARCUT only at target site, and resultant specific overhangs were blunted by treatment with Mung Bean Nuclease and Klenow fragment. This fragment having blunt ends at both termini was ligated with the gene of enhanced green fluorescent protein (EGFP) prepared by PCR. The resultant recombinant vector was translated and the fluorescent protein was successfully expressed in cells. Throughout the manipulation, neither natural restriction enzymes nor homologous recombination was employed. Thus, this strategy has promising applicability to construction of still larger vectors.

Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

An alternative approach to the use of antibiotic selection markers for maintenance of recombinant plasmid vectors in Escherichia coli based on an aminoacid auxotrophy complementation has been developed. An E. coli M15-derivated glycine-auxotrophic strain of has been constructed by means of a PCR-based approach. This mutant strain contains a deletion in the glyA gene, which encodes for serine hydroxymethyl transferase, an enzyme involved in the main glycine biosynthesis pathway in E. coli. Also, we have constructed the complementation plasmid pQEαβrham derived from the commercially available expression vector pQE40 (QIAGEN) containing the glyA homologous gene under the control of the constitutive weak promoter P3. By using the E. coli M15ΔglyA strain combined with the pQEαβrham plasmid, a successful complementation system was achieved, allowing transformants to grow on minimal media without glycine supplementation. The capability of the new system E. coli M15ΔglyA/pQEαβrham for recombinant overproduction of rhamnulose 1-phosphate aldolase was evaluated in antibiotic free fed-batch cultures at controlled specific growth rate, obtaining high cell density cultures and high RhuA production and productivity levels comparable to those obtained with the conventional system. The new selection marker based on glycine-auxotrophy is a promising genetic tool, not only for recombinant protein production, but also for plasmid DNA production processes, where antibiotics can not be present in the medium formulation.

Expression of aquaporin-1 in rat pleural mesothelial cells and its specific inhibition by RNA interference in vitro

The discovery of water channel aquaporins (AQPs) has greatly expanded the understanding of the regulation of the water permeability of biological membranes. Aquaporin-1 (AQP1) may be involved in fluid transport in numerous pathological conditions. The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells (PMCs) and to investigate the specific inhibitory effect of RNA interference (RNAi) on AQP1 expression in PMCs, which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo. PMCs were isolated and cultured from rat pleura. The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). Two eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the AQP1 gene of rat sapien were designed and constructed. The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes. Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection. The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection. RT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs. Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. The levels of the expression of AQP1 were inhibited by 83.45%, 90.93%, respectively, at mRNA level and 41.24%, 67.60%, respectively at protein level by two recombinant plasmids at 48 hours after transfection. The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells (P < 0.01). There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific shRNAs (P < 0.05). The expression of AQP1 was present in rat PMCs. The application of shRNA-AQP1 could markedly inhibit the expression of AQP1 in cultured rat PMCs. The use of RNAi is a promising tool for future research into the mechanisms of pleural fluid in vivo.

Identification of the functional role of AF1Q in the progression of breast cancer

A novel highly metastatic MDA-MB-231HM cells, derived from MDA-MB-231, was established in our institute. RT-PCR, real-time PCR and Western blot showed that AF1Q gene was differentially expressed between highly metastatic MDA-MB-231HM cells and its parental MDA-MB-231 cells. However, its molecular mechanisms in breast cancer metastasis remain to be characterized. To investigate the effects of AF1Q on the progression of human breast cancer cells, in the present study, recombinant expression plasmid vectors of the human AF1Q gene was transfected into MDA-MB-231 cells. We demonstrated that AF1Q overexpression enhanced the in vitro proliferation and invasive potential of breast cancer cells. Focused microarray analyses showed that 22 genes were differentially expressed between AF1Q transfected cells and its parental counterparts. Integrin alpha3, accompanied by up-regulation of Ets-1 and MMP-2, significantly enhanced the in vitro invasive potential of human breast cancer cells mediated by AF1Q. Estrogen-responsive ring finger protein gene (EFP), also played a role in the enhancement of in vitro proliferation of human breast cancer cells mediated by AF1Q, accompanied by down-regulation of 14-3-3delta. The association was ERalpha independent. These results were further demonstrated by RNA interference (RNAi) experiment in vitro. In in vivo study, we also demonstrated that AF1Q transfected breast cancer cells grew much faster and had more pulmonary metastases than vector-transfected or its parental counterparts. On the contrary, AF1Q knockdown cells grew slower and had less pulmonary metastasis. Similar effects of AF1Q on integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression observed in vitro studies were also found in the in vivo study. Taken together, these results provide functional evidences that overexpression of AF1Q leads to a more progression in human breast cancer, at least in part, through regulating the integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression.

Identification of MSRA gene on chromosome 8p as a candidate metastasis suppressor for human hepatitis B virus-positive hepatocellular carcinoma

BackgroundThe prognosis of patients with hepatocellular carcinoma (HCC) still remains very dismal, which is mainly due to metastasis. In our previous studies, we found that chromosome 8p deletions might contribute to metastasis of HCC. In this study, we aimed to identify the candidate metastatic suppressor gene on chromosome 8p.MethodsOligo-nucleotide microarrays which included 322 genes on human chromosome 8p were constructed to analyze the difference in gene expression profiles between HCC tissues with and without metastasis. The leading differentially expressed genes were identified and selected for further analysis by real-time PCR and Western blotting. Recombinant expression plasmid vectors for each target gene were constructed and transfected into HCC cells and its in vitro effects on proliferation and invasion of HCC cells were also investigated.ResultsSixteen leading differentially expressed genes were identified from the HCC tissues with metastasis compared with those without metastasis (p < 0.01, q < 16 %). Among of the 10 significantly down-regulated genes in HCC with metastasis, methionine sulfoxide reductase A (MSRA) had the lowest p value and false discovery rate (FDR), and was considered as a potential candidate for metastasis suppressor gene. Real-time PCR and Western blotting confirmed that the mRNA and protein expression levels of MSRA were significantly decreased in HCC with metastasis compared with those without metastasis (p < 0.001), and MSRA mRNA level in HCCLM6 cells (with high metastatic potential) was also much lower than that of other HCC cell lines. Transfection of a recombinant expression plasmid vector and overexpression of MSRA gene could obviously inhibit cell colony formation (4.33 ± 2.92 vs. 9.17 ± 3.38, p = 0.008) and invasion (7.40 ± 1.67 vs. 17.20 ± 2.59, p= 0.0001) of HCCLM6 cell line.ConclusionMSRA gene on chromosome 8p might possess metastasis suppressor activity in HCC.