Construction of RHBDF2 gene over-expression lentivirus vector and establishment of RHBDF2 cell line stably expressing RHBDF2 / 吉林大学学报(医学版)

Objective: To construct the RHBDF2 gene over-expression lentivirus vector and to establish the KA. hy926 cells stably expressing RHBDF2, and to provide the evidence for the construction of RHBDF2 gene over-expression lentivirus vector and the establishment of RHBDF2 cells stably expressing RHBDF2. Methods: According to the sequence of RHBDF2 gene provided by NCBI, and the primers were designed and synthesized; the RHBDF2 gene was amplified by PCR method, and the target gene was cloned into the entry vector by Gateway cloning technology, and then subcloned into the lentivirus vector pLV [Exp]-EGFP to construct the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2; the lentivirus expression vector plasmid pLV I Exp]-EGFP and the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2 were co-transfected into the HEK293T cells with the virus-assisted packaging plasmids to package the lentivirus and the titer of the lentivirus was detected. The EA. hy926 cells infected with pLV [Exp]-EGFP-control were used as control group and the EA. hy926 cells infected with pLV [Exp]-EGFP-RHBDF2 were used as experiment group. The EA. hy926 cells stably expressing RHBDF2 were screened by puromycin. The fluorescent quantitative PCR (qPCR) and Western blotting methods were used to detect the expression levels of RHBDF2 mRNA and protein in the EA. hy926 cells in control group and experiment group. Results: The enzyme digestion electrophoresis and sequencing results showed that the gene sequence of the EA. hy926 cells over-expression lentivirus vector in experiment group was completely consistent with the designed and synthesized sequence. The lentivirus titer in control group was 1 X 10 TU • mL , and the lentivirus titer in experiment group was 3X 10 TU • mL . The EA. hy926 cells were successfully infected with the lentivirus under fluorescence microscope and the infection efficiency was above 95%. The qPCR detection results showed that the expression level of RHBDF2 mRNA in the EA. hy926 cells in experiment group was higher than that in control group (P'<0.01). The Western blotting results showed that the expression level of RHBDF2 protein in the EA. hy926 cells in experiment group was higher than that in control group ( P < 0. 05 ). Conclusion: The lentivirus vector over-expressing RHBDF2 is successfully constructed∗ and the EA. hy926 cell line stably up-regulating the expression of RHBDF2 is established by using pLV [Exp]-EGFP-RHBDF2 lentivirus.

Effects of silencing the HIF-1α gene on the expression of BSP and osterix in rat BMMSCs under tension / 口腔疾病防治

Objective@#To explore the effect of hypoxia inducible factor 1α (HIF-1α) gene silencing in rat bone marrow mesenchymal stem cells (BMMSCs) under mechanical distraction on the expression of bone sialoprotein (BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs.@*Methods @#The shRNA sequence was designed according to the rat HIF-1α gene, and the pGMLV-SC1RNAi lentiviral vector was cloned after PCR amplification. After screening positive clones and identifying competent transformed cells by sequencing, 293T cells were packaged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF-1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF-1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF-1α protein in the four groups to verify the response of the target genes and exclude off-target effects. A Flexcell FX-5000T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT-PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF-1α shRNA group, and negative control group.@*Results@#The HIF-1α shRNA lentiviral vector was successfully constructed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the response and the blank control group (P > 0.05). The recombinant lentivirus could effectively silence HIF-1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF-1α shRNA group showed significantly increased mRNA and protein expression of the bone-related factors BSP and osterix (P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups (P > 0.05).@*Conclusion @#Silencing HIF-1α in BMMSCs under mechanical distraction can promote the expression of BSP and osterix.

Construction of lentivirus-mediated mito-OGG1 gene overexpression and OGG1 gene knockdown model in 661W cells / 中华实验眼科杂志

Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells.Methods Constructed the target plasmids,including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ (COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot.Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0× 107to 6.0× 107 TU/ml.The optimal MOI of 661W cells was 40,and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100％ after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group,OGG1 group,overexpression control group,shRNA group and low expression control group were 1.000±0.000,41.581±12.206,0.888±0.056,0.239±0.121 and 1.081±0.083,and the relative expression levels of OGG1 protein were 1.029±0.153,1.657 ± 0.237,0.752 ± 0.143,0.471 ± 0.149 and 1.036 ± 0.185,respectively,with significant differences between them (F=44.654,30.948;both at P＜0.05),the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group,the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group,with significant differences between them (all at P＜0.05).Conclusions The mitoOGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed,which provides the preliminary experimental basis for follow-up study.

Transfection with lentivirus carrying Ifi204 genes up-regulates p204 expression of the aortic vascular fibroblasts in rats / 基础医学与临床

Objective To observe the effect of transfection with lentivirus carrying Ifi204 genes on the expression of p204 in rat vascular adventitial fibroblasts. Methods Culturing the vascular adventitial fibroblasts of rat aortic and then conducting immunocytochemistry to identify the type and purity of these rat fibroblasts. The vascular adventitial fibroblasts of rat aortic cultured were transfected with lentivirus vector system carrying Ifi204 genes (Ifi204 group), the fibroblasts which has been transfected with the empty vector lentivirus particles as the control group (group C), and the untreated fibroblasts as the blank group (group N). The expressions of mRNA and protein of p204 in the fibroblasts were detected by realtime fluorescent quantitative PCR and Western blot test separately. Results There was expression of p204 in group N. Compared with group N and group C, mRNA and protein expression of p204 in the Ifi204 group had been all upregulated (P＜0.01). Conclusions There is structural expression of p204 in vascular adventitial fibroblasts of rat aortic. Transfection with lentivirus carrying Ifi204 genes can upregulate the expression of p204 in vascular adventitial fibroblasts.

Preparation of DJ-1 overexpressing cell model by using lentiviral vector / 基础医学与临床

Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166P mutant, and to investigate the role of lentiviral vector in gene overexpressing cell model .Methods Wild type DJ-1 and L166P mutant DJ-1 lentiviral vector plasmids were respectively constructed .After sequencing and comparing cor-rectly, the plasmid was amplified and transfected into HEK 293T cell line.Expression of WT DJ-1 and L166P mu-tant DJ-1 in cell lines was detected by fluorescence and Western blot .After determining the accurate expression of the target protein, a large amount of HEK293T cells was transfected and packaged to produce lentiviral particles. The PC12 cells were infected with the titer of virus supernatant.The fluorescence intensity of GFP and the expres-sion of target protein were observed by fluorescence microscope and Western blot method ,and the infection effi-ciency of the virus was determined .Results Lentiviral vectors carrying wild type DJ-1 and its mutants were suc-cessfully constructed .The virus vector can be transfected into HEK 293T cells and the target protein can be correctly expressed.The viral titers of LV-DJ-1 and LV-DJ-1/L166P were 2×109 TU/mL and 2×108 TU/mL, respectively. Virus supernatant can efficiently infect PC 12 cells, and most cells can express target proteins .The protein expres-sions of exogenous wild-type DJ-1 and L166P mutants were 315% and 285% of endogenous content ,respectively. Conclusions Lentivirus vector can infect cells efficiently , and it is a good way to prepare gene over expressing cell model.A cell model overexpressing DJ-1 or its L166P mutant is successfully prepared .The model can be used for subsequent DJ-1 function research .

Construction and the infection efficiency of BimS lentivirus RNA interference vector / 实用口腔医学杂志

Objective:To construct BimS lentivirus RNA interference(RNAi) vector and to study its infection efficiency by using RNAi technique.Methods:Three interference targets were designed according to the BimS sequence.The single chain primer was annealed into double-stranded oligo sequences,and then connected with vector linearized with Age Ⅰ and EcoR Ⅰ enzyme.The recombinant plasmid was packaged,and the infection efficiency was observed by infecting ACC-2 cells.Results:After amplification,a 337 bp band was appeared in the electrophoresis results of positive clones.Sequence of inserted fragments were identical with the result of DNA sequencing.Restructuring lentivirus was packed in 293T cells,the virus titer was 2 × 108 TU/ml,MOI =20,and the transfection efficiency was 85％.The BmS mRNA relative expression of pFU-GV-BmS-1,pFU-GV-BMS-2 and control group was 0.743 ±0.025,0.466 ±0.023 and 1.266 ±0.042 respectively(between each 2 groups,P ＜0.05).Conclusion:BimS lentivirus virus RNA interference vectors can be constructed,and can efficiently infect ACC-2 cells.

Construction of VEGF165 lentivirus vectors and expression in adipose tissue-derived stem cells after transfection / 中国药学杂志

OBJECTIVE: To clone target gene by RT-PCR method, construct VEGF165 lentivirus vectors, transfect adipose tissue derived stem cells (ADSCs) and verify the expression of VEGF165 in vitro and in vivo. METHODS: RT-PCR technology was employed to clone VEGF165 gene, and this gene was cloned to lentivirus vector pLVX-EF1α-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1α-VEGF165-IRES2-AcGFP1. Bacterial colonies PCR and sequencing analysis were used for identification. Then, Lenti-X 293T cells were transfected with main vector pLVX-EF1α-VEGF165-IRES2-AcGFP1, packaging plasmid gag-pro, vpr-pol, Tet-Off™, tat-IRES-rev and coating plasmid env (VSV-G). Lentiviral vectors were packaged and the titer was determined. ADSCs were isolated by collagenase digestion method, then cultured, and identificated by morphology, immunofluorescence and multi-directional differentiation. ADSCs was transfected with packaged VEGF165 lentivirus. Immunofluorescence and ELISA were used to detect the expression of VEGF165 in vitro. ADSCs transfected with VEGF165 lentivirus were injected into nude mice. ELISA was used to detect the expression of VEGF165. RESULTS: The VEGF165 gene fragment was cloned successfully, and the lentiviral vector plasmid pLVX-EF1α-HVEGF165-IRES2-AcGFP1 was confirmed to contain VEGF165 gene fragment as shown by bacterial colonies PCR. DNA sequencing analysis confirmed that VEGF165 gene sequencing was exactly the same with that reported by Genbank. After transfection, a large number of Lenti-X 293T cells with green fluorescence were observed by fluorescent microscopy. The concentration of the virus titer was 1×108·TU·mL-1. ADSCs were identified by morphology, immunofluorescence and multi-directional differentiation methods, in line with the literature reported ADSCs characteristics. There were about 90% of ADSCs which could express VEGF165 after being transfected with the viruses by immunofluorescence detection, also, VEGF165 protein was proved by ELISA to express stably and efficiently in vitro and in vivo. CONCLUSION: Lentiviral vectors expressing VEGF165 are successfully constructed by cloning target gene with RT-PCR method. After transfection, ADSCs expressing VEGF165 protein stably in vitro and in vivo can be obtained.

The Comparative Study of Different Methods for Gene Transfer to Cochlear Hair Cells / 听力学及言语疾病杂志

Objective To obtain an easy and high efficient method for gene transfer to cochlear hair cells ,by comparing three mediating green fluorescent protein (GFP) methods (electroporation ,adenovector and lentivirus vector) .Methods Cochlear sensory epithelium was dissected from anaesthetized P2 mice .Sensory epithelia were transferred onto poly -L -lysine treated cover slides and cultured overnight .Gene transfer was performed by elec‐troporation in medium containing pGPHI/GFP/Neo plasmid or by incubation with diluted recombined adenovirus/lentivirus vector .After 48 hours ,green fluorescence was checked under fluorescence microscope .To confirm the ef‐ficiency of exogenous gene transfer ,real-time PCR was performed using specific primers .Results The transfec‐tion efficiencies of electroporation and lentivirus vector mediated gene transfer were very low .Both immunofluores‐cence and real - time PCR results showed that the transfection efficiency of adenovirus mediated GFP and Bmi1 transfer were relatively higher .The proportion of GFP positive cells in outer hair cells and inner hair cells of middle turn were 90 .0% ± 4 .1% and 5% ± 0 .4% ,respectively .Conclusion Adenovector is more efficient for exogenous gene transfer to cochlear hair cells ,thus adenovector is a good carrier for gene transfer to cochlear hair cells .

Construction and identification of a recombinant lentivirus harboring expressing Mus musculus STAT3 gene / 重庆医学

Objective To construct recombinant lentivirus with the gene STAT3 of the Mus musculus,measure the expres-sion of STAT3,and conduct lentivirus packing and identification.Methods The mRNA of mouse myoblast was extracted and transformed into STAT3 cDNA by the special primer.then,STAT3 cDNA was amplified and reclaimed and inseted into pLVX-IRES-ZsGreen1 vector.Cleavage map and sequencing analysis were used for identification of the recombinant lentivirus vector (pLVX-IRES-ZsGreen1-STAT3).293 T cells were transfected with main vector pLVX-IRES-ZsGreen1-STAT3.and 48 h later, Western blott detected the expression of STAT3 protein.Lentiviral vectors were packaged and the titer was determined.Results The lentiviral vector plasmid pLVX-IRES-ZsGreen1-STAT3 was identified correctly by cleavage map and Co-transfection of 293 T cells with 48 h,the expression of STAT3 was significantly enhanced by western blot.And DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by genbank.Conclusion Lentiviral vector carrying STAT3 was successfnlly constructed and could express STAT3 with high efficiency,and can be used in further study.

Construction of lentiviral vector of peroxiredoxin 2 gene mediating RNAi and its effects on the proliferation of SW480 cell / 重庆医学

Objective To construct a lentiviral expression vector of peroxiredoxin2(PRDX2) RNA interference (RNAi) and to investigate the effect of siRNA of PRDX2 genes on the proliferation of human colonrectal cancer SW480 cell .Methods RNAi tar‐get sequences were designed and synthesized towards the PRDX2 gene sequences .The lentiviral vector pGC‐EGFP‐shPRDX2 was constructed and identified .The vector was transformed into SW480 cells ,and the transfection efficiency was evaluated by fluores‐cence microscopy .The expression of PRDX2 was detected with Quantitative real‐time PCR (qRT‐PCR) and Western blot in the transfected cells .Cell growth and colony forming ability were detected with MTT and plate cloning technique .Results PRDX2 gene lentiviral vector was successfully established and was proved by gene sequencing .The expression of PRDX2 in mRNA and pro‐tein was significantly reduced(P<0 .05) .The PRDX2 mRNA and protein expression in SW480 transfected with lentiviral were sig‐nificantly reduced (P< 0 .05) ,and the ability of growth and proliferation were significantly reduced(P< 0 .05) .Conclusion PRDX2 gene lentiviral vector could be a stable and reliable tool .The proliferation and growth of SW480 cells transfected by pGC‐EGFP‐shPRDX2 could be effectively suppressed ,which could facilitate further investigation of the roles of PRDX2 gene in the de‐velopment and progression of colorectal cancer .

Construction of JSRV-NM Pseudovirions by High Efficiency Packaging System of the Lentivirus / 天津医药

Objective To overcome the fact that SRV-NM virus can only multiple and amplify through partially pu-rified jaagsiekte retrovirus inoculated intratracheally in sheep but it cannot be augmented using in vitro cell culture, we con-structed JSRV-NM pseudovirions based on high efficiency packing system of lentivirus. Methods Lentivirus of three high efficiency packing plasmids system pMD.G, pCMV-HIV 8.2 and pHIV-eGFP was developed, and JSRV-NM-env coated plasmid pCMVJSRV-NM was used to substitute VSV-G virus coated plasmid pMD.G then co-transfected into 293T cells to replicate, package and produce restructured JSRV-NM pseudovirions. Gene expression of pseudovirion was determined through WPRE using real time PCR; Virus infectivity was detected through inoculating JSRV-NM pseudovirions into 24 pore plates. Results We construct JSRV-NM pseudovirions successfully based on the lentivirus system. JSRV-NM pseudo-virions can also be concentrated to higher titer (108 TU/mL detected by real time PCR by ultracentrifugation without signifi-cant loss of activity. JSRV-NM and VSV-G pseudovirions infected on Hela cells (both MOI= 3) respectively and no obvi-ous difference were shown on their infection efficiency detected by real time PCR. Conclusion Based on lentivirus system, JSRV-NM pseudovirions can be multipled and amplified in 293T cell culture in vitro. JSRV-NM pseudovirions is stable without loss its infection activity and the requirements of biological laboratory safety II was also met. JSRV-NM pseudoviri-ons will provide a useful tool for further study of JSRV-NM-env infection across species or its induction of lung adenocarci-noma.

Rat ADSCs transfected by lentivirus vector-mediated enhanced green fluorescent protein / 中华显微外科杂志

Objective To explore the labeling method of rat adipose-derived stromal cells,and observe the stem cell characteristics and the activities of EGFP-positive adipose-derived stromal cells (EGFP-ADSCs) in vitro and in vivo.Methods ADSCs were transfected for 12 h with enhanced green fluorescent protein gene (EGFP) carried by lentivirus(Lv-EGFP) vector at different value of MOI (0,5,10,25,50,100,respectively).The rate of EGFP expression and fluorescence intensity were evaluated by flow cytometric analysis and fluorescence microscopy,and cell viability was detected by MTT-test after transfection.Secondly,cells were exposed either to adipogenic medium or osteogenic medium,then stained with Oil Red O and Alizarin Red S.Cell growth was investigated on frozen longitudinal sections when EGFP-ADSCs were injected into acellular nerves to build tissue-engineered peripheral nerves repairing sciatic nerve defects in rats for 1 week in vivo.Results EGFP-positive rate and fluorescence intensity peak at 4 days after transfection.The rate of EGFP expression was 0.13％,31.09％,75.33％,92.66％,96.70％,98.38％ for MOI =0,1,5,25,50,100,respectively.The positive rate between the experimental group and control (MOI =0) existed significantly difference (P ＜ 0.05) ; the difference between MOI =1,5 groups and MOI =25,50,100 groups were also observed (P ＜ 0.05).There was no statistical difference in EGFP-positive rate and cell proliferation activity among MOI =25,50,100 groups (P ＞ 0.05).MOI =25 was chosen as best scheme to transfect ADSCs for subsequent experiments.Osteogenic and adipogenic differentiation for 20 days,orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red O staining.At 1 week in vivo,EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections.Conclusion Lv-EGFP transfection does not affect the ADSCs activity and their osteogenic and adipogenic differentiation,so could be as a tracing method for ADSCs-tissue-engineered peripheral nerves repairing nerve defects.

Speciifc targeted-intervention effect of CXCR 7-shRNA mediated by low viral carrier plus Rhizoma Paridis total saponin on tumor cells:an experimental study / 中国生化药物杂志

Objective To investigate the CXCR 7 protein expression when CXCR 7-shRNA transfected into human gastric cancer cell which mediated with lentivirus vector combined with Rhizoma Paridis Total Saponin. Methods Three shRNA sequences of CXCR 7 and one negative control sequence were designed and synthesized, and recombinant lentiviral vectors with pSilencerTM 4.1 system were established. Transfection of HEK 293 T cells and packaging viral were finished and the titers were detected. Transfection of all recombinant lentiviral vectors and negative control vector were finished and expression of CXCR 7 mRNA were detected by RT-PCR method. Silence efficiency in groups were determined and the expression vector with highest silence efficiency was selected for next experiments. To detect the effect of SGC 7901 cell proliferation by CXCR 7-shRNA transfection and combined with Rhizoma Paridis Total Saponin intervention with MTT. To detect the effect of SGC 7901 cell expression of protein by CXCR 7-shRNA transfection and combined with Rhizoma Paridis Total Saponin intervention with Western blot. Results The packaging of three lentiviral vector and negative control sequence are successful which is confirmed by gene sequencing and the titer are 4.9×108 pfu/mL, 3.6×108 pfu/mL, 5.2×108 pfu/mL, 2.0×108 pfu/mL respective. The expression quantity of CXCR 7 mRNA in positive groups are lower than negative control group(P<0.05)and inhibition ratio to CXCR 7 in CXCR 7-shRNA-1 and combined with Rhizoma Paridis Total Saponin intervention group is higher than the other two groups(P<0.05). The proliferation level of tumour cell is significant reduction after CXCR 7-shRNA-1 transfection and have a significant difference comparing to the group without transfection(P<0.05). The expression of CXCR 7 protein is significant reduction after CXCR 7-shRNA-1 transfection comparing to the group without virus vector and negative control group and have a significant difference(P<0.05). Conclusion The construction of three CXCR 7-shRNA lentiviral expression vector are successful and expression level of protein and CXCR 7 mRNA are down-regulated effectively after transfection and combined with Rhizoma Paridis Total Saponin intervention. It maybe means that CXCR 7 gene takes an important role in the process of gastric cancer proliferation and invasion.This is foundation for further study of gastric cancer gene therapy using CXCR 7/CXCL 12 biological axis as a target.

Construction of lentiviral vector-mediated siRNA knockdown of ER-α36 and its action on gastric cancer cell growth / 中国病理生理杂志

[ ABSTRACT] AIM:To construct a lentiviral vector for stable delivery of the ER-α36 gene and to detect its effect on SGC7901 cell growth.METHODS: The efficient RNAi targeting sequences identified for the ER-α36 gene were screened.The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA.Then it was digested by Xho I and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector.PCR was used to screen the positive clones and sequence.The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line.Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect.17β-estrodial at concentration of 1 ×10 -10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined.RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors.Immunofluorescence assay demonstrated that transfection efficiency was above 80%.Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mR-NA and protein levels with tetracycline ( TeT) simulating as revealed by real-time PCR and Western blotting.Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silence ER-α36 expression are con-structed successfully and can be used to study the role of ER-α36 in gastric cancer.The ER-α36 is related with many kinds of cancer cell growth, including gastric cancer cells.

Construction and identification of RNAi lentiviral vectors targeting human midkine gene / 临床与实验病理学杂志

Purpose To construct a lentiviral ventor-mediated RNA interference of human midkine ( MK) gene and to provide the ba-sis for further experiment in vivo and in vitro. Methods Four RNAi sequences targeting human MK gene were designed, and cloned into the lentiviral vector to construct lentiviral vectors:GV115-MK-1, GV115-MK-2, GV115-MK-3 and GV115-MK-4. After transfec-tion into competent E. Coli bacteria, the candidate clones were identified by PCR and DNA sequencing. The titer of lentiviruses was determined after 293 T cells were co-transfected with GV115-MK, pHelper 1. 0 and pHelper 2. 0. The four kinds of recombinant lenti-viruses were used to infect human breast cancer cell MDA-MB-231, and the expression levels of MK mRNA were detected by real-time PCR. Results PCR analysis and DNA sequencing confirmed that the four inserted MK RNAi sequences were corrected. Strong green fluorescence was observed in the 293 T cells under the fluorescent microscope after co-transfection. The titer of the concentrated virus was 6 × 108 , 5 × 108 , 5 × 108 and 6 × 108 TU/ml, respectively. The MK expression in MDA-MB-231 cells infected with lentiviral vec-tors GV115-MK-1 was decreased by 87. 2% (P<0. 05). Conclusion Lentiviral RNAi vector for MK gene is successfully construc-ted, and it could effectively inhibit the expression of MK gene in MDA-MB-231 cells in vitro.

Construction of a lentivector containing over-expressing β-catenin gene by multisite Gateway technology / 国际生物医学工程杂志

Objective To construct a lentiviral vector over-expressing β-catenin gene by multisite Gateway technology and confirm its effect.Methods By using multisite Gateway clone technique,the entry clone of pDown-Ctnnb1 was constructed using BP recombination reaction.Then,LR recombination reaction was performed among pUp-EF1A,pDown-Ctnnb1,pTail-IRES/DsRed-Express2 and pLV.Des3d.P/puro to generate an expression clone of pLV.EX3d.P/puro-EF1A＞Ctnnb1 ＞IRES/DsRed-Express2.In each step,PCR and sequencing analysis were used to verify the constructions.When it was verified that plasmids were transfected into 293T cells,PT-PCR was performed to determine the mRNA level of β-catenin gene.Results Both PCR and sequencing analysis revealed that β-catenin over-expression gene was inserted into the target site and the insertion sequence was perfectly corrected.The RT-PCR results showed that the expression of β-catenin gene was significantly upregulated.Conclusion The lenvivirus-mediate β-catenin over-expression gene was successfully constructed..

Construction of lentiviral vector of mFVII/Fc fusion gene and the research of its expression in human bone mesenchymal stem cells / 中华泌尿外科杂志

Objective To construct a recombinant Ientiviral vector of mFVII/Fc and investigate its transfective efficiency into human bone mesenchymal stem cells (hBMSCs),and to detect the expression of mFVII/Fc fusion gene in vitro. Methods Coagulation factor VII (FVII) was cloned in vitro,with a point mutation from Lys to Ala in the position of 341 in the gene level.The cDNA fragments of mutational FVII (mFVII) and those of IgG1Fc were fused together with DNA ligase.After digestion,integration and sequencing,the fusion DNA was identified and transfected human embryonic kidney 293T cell packaging for re-mFVII/Fc lentiviral vector.After successful identification of vectors,detect the Ientiviral titer determination,bulk transfer after the determination of best MOI value of the third generation of hBMSCs,obseve the GFP expression with fluorescence microscope,have relative quantitative analyse of mRNA and protein expression of mFVII/Fc with RT-PCR and ELISA at different time points. Results In contrast with GenBank ID: AF 272774,the fusion gene matches exactly except the synonymous mutation,and the titer of packaging lentivirus was 2×108 TU/ml.Analyzed by Flow cytometry, indentification results of hBMSCs were as follows,CD+29(98.08%),CD+44 (97.63%),CD+34(0.31%) and CD+45(0.58%),respectively.The transfection efficiency of hBMSCs after 72 hours was (84±3)%,and the hBMSCs with mFVII/FC transfcetion have a large number of mRNA transcription and protein expression levels. Conclusions In this experiment we obtained a stable genetic vector with hBMSCs fusion gene expression successfully,which lay a foundation for the tissue factor study of prostate cancer targeting therapy and cancer gene therapy research.

IL-10 overexpression attenuated expression of TNF-α and IL-1β activated by lipopolysaccharide in astrocytes / 中南大学学报(医学版)

Objective To investigate the intervention effect of lentivirus expressing human IL-10 (LV-hIL-10) on activated astrocytes.Methods DI TNC1 cell line was treated with different concentrations of lipopolysaccharide (LPS) and time points.The expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukinb-1β (IL-1β) was detected by RT-PCR and ELISA.Moreover,the effect on the expression of proinflammatory cytokines TNF-α and IL-1β was analyzed in DI TNC1 cell lines infected with and without LV-hIL-10.Results The expression of TNF-α and IL-1β was increased in DI TNC1 induced by LPS.The expression of IL-10 was upregulated in DI TNC1 infected with LV-hIL-10.TNF-α and IL-1β were inhibited by IL-10 overexpression in DI TNC1 actived by LPS.Conclusion DI TNC1 is activated by LPS and secretes proinflammatory cytokines TNF-α and IL-1β as immune-like cells,and these activation is inhibited by hIL-10 overexpression.

Establishment of FSP27 Gene Knockdown Preadipocyte Cell Line / 生物化学与生物物理进展

Obesity and its related metabolic diseases become major health problems in the world.Adipose tissue plays an important role in the development of obesity.FSP27,a member of the CIDE family proteins,is expressed at high levels in white adipose tissue and differentiated 3T3L1 cells.The objective of current study is to establish a FSP27 knockdown preadipocyte cell line to investigate the in vivo function of mouse FSP27.The double strand siRNA of mouse FSP27 corresponding to nucleotides 270 to 291 was synthesized and inserted into pSilencer2.1.pSilencer-siFSP27 was co-transfected into 293T cells with the HA-mFSP27 expression vector to test its knock-down efficiency.The FSP27 siRNA was then transferred to a lentiviral vector.Lentivirus were generated and used to infect 3T3-L1 cells.It was shown here that lentivirus containing FSP27siRNA can effectively knockdown FSP27 expression in 3T3-L1 cells.Establishment of FSP27 knock-down cell line provides a useful tool for the study of in vivo function FSP27.

Construction of a lentiviral expression vector that inhibits enhanced green fluorescent proteins / 医学研究生学报

Objective: To construct a lentiviral expression vector that inhibits enhanced green fluorescent proteins(EGFP).Methods: The EGFP shRNA was cloned into the pLL3.7 lentivirus vector digested with XbaⅠ/XhoⅠ.J558L and Hela cells were infected by lentiviruses,and the EGFP expression was observed with the fluorescence microscope.Results: The EGFP RNAi sequence was correctly cloned into the lentivirus vector with the U6 promoter,and the EGFP expressions of Hela and J558L cells were suppressed after infected by lentivirus containing EGFP shRNA. Conclusion: The lentivirus vector effectively inhibited EGFP expression,which could be used to mediate further researches on gene function.