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To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
Subject(s)
Humans , CRISPR-Cas Systems/genetics , Gene Editing , Gene Knockout Techniques , HCT116 Cells , /metabolismABSTRACT
Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.
Subject(s)
Tissue Plasminogen Activator , Process Optimization , Flow Cytometry/methods , Fluorescence , Cell Count/instrumentation , Clone Cells/classification , Plasminogen Activator Inhibitor 1/adverse effects , Green Fluorescent ProteinsABSTRACT
Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.
Subject(s)
Human bocavirus , Promoter Regions, Genetic , Transcriptional Activation , Viral Nonstructural Proteins , Virus ReplicationABSTRACT
Aim To construct different over-expression vectors of ST8Sial gene and establish a cell line with stable ST8Sial over-expression , and to check the proliferation of cells with ST8Sial over-expression. Methods Polymerase chain reaction ( PCR) was used to amplify the code region of ST8Sial. The amplified ST8Sial fragment and pEGFP-Cl vector, pHBLV-CMV-MCS-3flag-EFl-ZsGreen-T2A-Puro vector were digested with restriction enzymes. The target gene fragment and the different vectors were ligated to obtain the ST8Sial-pEGFP-Cl recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors. Melanoma cells WM451 were transfected with different vectors. Cell line with stable ST8Sial over-expressing was established. ST8Sial mRNA level was checked by real-time PCR. STSSial protein expression level was checked by Western blot. Proliferation of the stable cells was assayed by CCK-8 method. The clony formation of stable cells was also performed. Results Both ST8Sial-pEGFP-Cl recombinant plasmid and STSSial over-expression lentiviral vectors were successfully constructed. The transfection efficiency of ST8Sial over-expression lentiviral vector was much higher than that of ST8Sial-pEGFP-Cl recombinant plasmid. A WM451 cell line with stable ST8Sial over-expression lentiviral vectors was established. Results showed that the over-expression of ST8Sial promoted the proliferation and colony formation of cells. Conclusions ST8Sial over-expression vectors are successfully constructed. The over-expression of ST8Sial promotes the proliferation and colony formation of WM451 cells.
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Objective Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of malignant kidney tumors and its pathogenesis has not been elucidated. Our previous studies showed a positive correlation of Glucose-6-phosphate dehydrogenase (G6PD) with the development, progression and poor prognosis of ccRCC. In this study, we first established a G6PD defect ccRCC stable cell line, detected the influence of G6PD knockdown on ccRCC migration, and provided a cell model for further studies on the functional and molecular mechanisms of G6PD in ccRCC.Methods Using the OligoEngine RNAi software, we designed siRNA targeting the human G6PD gene 3′ non-coding region and negative control siRNA sequences, inserted the double-stranded siRNA into the pSR-GFP/Neo expression vector through Bgl Ⅱ and Hind Ⅲ enzyme loci, and constructed Caki-1-G6PD siRNA and Caki-1-negative control cell lines, followed by transfection and G418 screening of the Caki-1 cells. We measured the expression and enzyme activity of G6PD in the cells by real-time RT-PCR, determined the cell migration phenotypes by Transwell assay, and detected the expressions of p-STAT3 and STAT3 by Western blot.Results Morphologically normal Caki-1-G6PD siRNA and Caki-1-negative control cells were seen under the fluorescence microscope. With GFP expression as a marker, the transfection efficiency rate of the cells was 45－55%. The density of the adherent cells at 48 hours was 90% and their transfection efficiency rate was over 60%. Compared with the Caki-1-negative control cells, the Caki-1-G6PD siRNA cells showed significant decreases in the expressions of Caki-1-G6PD mRNA and protein (P<0.01), enzyme activity (P<0.05), relative count of migratory cells (64.0±4.2 vs 30.0±2.9, P<0.01), and the ratio of p-STAT3/STAT3 (0.45±0.05 vs 0.24±0.01, P<0.01).Conclusion The Caki-1-G6PD siRNA cell line with stable G6PD knockdown and a lower migration ability was first successfully constructed, and the decreased migration ability induced by G6PD knockdown is associated with the STAT3 signal, which is contributive to an insight into the functional and molecular mechanisms of G6PD in the development and progression of ccRCC as well as to finding intervention targets for the treatment of ccRCC.
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To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.
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Objective To optimize multiplicity of infection ( MOI) and antibiotics ( blasticidin) concentration selecting BSD gene in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2.Methods The INS-1 cells were transfected by fluorescence labeled negative control SGMS2-siRNA lentivirus at MOI of 0, 10, 30, 60 and 120 TU number/cell.The cells were photographed under fluorescent microscopy after 72 h cultivation, then fluorescence ratio and apoptosis rate were calculated to determine optimal MOI.The INS-1 cells were treated by blasticidin with different concentrations of 0, 1, 2, and 3 μg/mL, and the apoptosis rate was observed to acquire optimal concentration of antibiotics.The INS-1 cells were transfected by negative control SGMS2-siRNA lentivirus and SGMS2-siRNA lentivirus (virus titer:1 ×108TU/mL) at optimal MOI and positive-transfected cells were selected by blasticidin at optimal concentration, then mixed cell lines were acquired.The monoclonal cell line was constructed at fluorescence ratio of 90%.Results The optimal MOI was 60 with 100% fluorescence ratio, less than 0.5% apoptosis rate and keep original cellular morphology.The optimal concentration of blasticidin was 2 μg/mL with cell adherence disappear and all cells apoptosis.The Ct value of INS-1-SEMS2 cells detected at the second time was 28.21, which was greater than 27.58 at the first time.The interfering efficiency of siRNA was 77.78% which indicated a successful expression of siRNA and construction of monoclonal stable cell line ( INS-1-SEMS2 ).Conclusion The monoclonal stable cell line was successfully constructed by lentivirus vector-mediated RNA interence silenced gene SGMS2.
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Aim To construct the stable hFcεRIα/RBL-2H3 cell line expressing human FcεRIα( hFcεRIα) . Methods The human FcεRIα gene was obtained by RT-PCR and cloned into the eukaryotic expression vector pCI-neo. Then, the pCI-neo-hFcεRIα vector was transfected into RBL-2H3 cells by lipo-somes, and the transfected cells were screened through G418 fil-tration subsequently. Finally, RT-PCR, Western blot and immu-nofluorescence assay were used to determine the result of trans-fection. Results According to the optimized transfection param-eters, the transfection efficiency reached 75. 38%. The results of Western blot, immunofluorescence and RT-PCR showed that hFcεRIα could be expressed in RBL-2H3 cells successfully. Conclusion HFcεRIα/RBL-2H3 cells were successfully con-structed,which will be the experimental basis for further study on the mechanism of IgE/FcεRI and drugs for allergy diseases.
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Background and purpose:Overexpression of inhibitor of protein phosphatase 2 A-2 (I2PP2A) in many tumors including gastric cancer suggests that I2PP2A may contribute to the development of gastric cancer. To further study the biological function of I2PP2A and its role in gastric cancer, we established a BGC823 cell line for stable expression of shRNA targeting human I2PP2A gene. Methods: A double-stranded shRNA targeting the I2PP2A was designed, synthesized and was inserted into a lentivirus vector (pGLV2), and the insertion was identiifed by restriction endonuclease analysis and DNA sequencing. BGC823 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with puromycin. The expression of I2PP2A was examined using real-time PCR (RT-PCR) and Western blot. Results:Sequencing result proved that recombinant lentivirus vector pGLV2-shRNA-I2PP2A was constructed correctly. RT-PCR and Western blot results conifrmed that the expression of I2PP2A was signiifcantly down-regulated in this infected BGC823 cell line. The efifciency of siRNA interference of I2PP2A could be up to about 90%. Conclusion:A lentiviral vector carrying a shRNA targeting the I2PP2A gene is successfully constructed, and a BGC823 cell line stably expressing I2PP2A shRNA is established with this lentiviral system.
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Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.
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OBJECTIVE@#To study the expression of TRPC6 among prostate cancer cells, establish high expression cell lines of TRPC6, and to provide potential cell mode for prostate cancer oncogenesis and development.@*METHODS@#Occurrence and development of prostate cancer cells, PC3, PC-3 m DU145, 22 rv1, LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method. Calcium phosphate transfection method was used to package retrovirus pLEGFP-N1-TRPC6 and pLEGFP-N1-vector and infect the prostate cancer cells, a stable high expression of TRPC6 prostate cancer cells. Sable cell lines of TRPC6, matrix metalloproteinase (MMP) 2, MMP9 expression was detected by QPCR and Western blot. Change of cell invasion ability was detected by Transwell.@*RESULTS@#The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells. Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest, and high transfer cell line PC-3M express was the highest. Real-time fluorescent quantitative PCR and western blot results showed that after filter, the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously. Transwell experimental results showed that the overexpression of TRPC6 could promote the invasion ability of PC3 prostate cancer cells.@*CONCLUSIONS@#TRPC6 expressed in prostate cancer cells is in disorder, and its action may be associated with the invasion and metastasis of prostate cancer cells; successful establishment of stable high expression of TRPC6 prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer, and lay down the Foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer mechanism.
Subject(s)
Humans , Male , Cell Line, Tumor , Matrix Metalloproteinase 2 , Genetics , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , Neoplasm Invasiveness , Genetics , Prostatic Neoplasms , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Retroviridae , TRPC Cation Channels , Genetics , Metabolism , TRPC6 Cation ChannelABSTRACT
OBJECTIVE@#To investigate the effects of miR-25-3p on the occurrence, development and proliferation of tongue squamous cell carcinoma cells.@*METHODS@#To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant retroviral vector-mediated gene transfer method. The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assays. cyclinD1, p21(cip1) and p27(kip1) mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR. cyclinD1, p21, p27(kip1), AKT, p-AKT, FOXO1 and p-FOXO1 expressions in the transfected Tca8113 were detected by western blot analysis. In addition, miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.@*RESULTS@#Quantitative PCR showed that miR-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue. MTT and cell colony formation assays showed that after miR-25-3p overexpression, the proliferation of transfected Tca8113 was obviously attenuated. Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression, p21(cip1) and p27(kip1) expressions were upregulated, while cyclinD1, AKT, FOXO1 expressions were downregulated, and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.@*CONCLUSIONS@#MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression, playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.
Subject(s)
Humans , Carcinoma, Squamous Cell , Genetics , Metabolism , Cell Cycle Proteins , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Gene Expression Regulation, Neoplastic , MicroRNAs , Genetics , Metabolism , Tongue Neoplasms , Genetics , Metabolism , TransfectionABSTRACT
Transient receptor potential A1 (TRPAl) is a cold sensitive cation channel, which could also be activated by various pungent compounds. As a transduction channel in a number of sensory modalities, TRPAl expressing in heterogonous systems serves to provide great convenience in pharmacological analysis and functional investigation. Due to cellular toxicity, establishment of stable TRPAl cell line has always been challenging. Nevertheless, the first stable human embryonic kidney (HEK-293) cell line with un-controlled expression of TRPAl was successfully established. It was also confirmed that this stable cell line retained TRPAl expression for more than 25 passages in culture. The functional analysis of the cell response verified the stability and specificity of this novel recombinant TRPAl cell line. Altogether, the data indicated this TRPAl-HEK cell line would be a useful tool for functional analysis of TRPAl and for the development of high throughput screening (HTS) compatible assay in the effort to identify TRPAl modulators.
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Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells.Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1.Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR,Real-time PCR and Western blot after transfection of the designed siRNA.Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay.Establishing NSPc1 stably knockdown cell line,comparing proliferation abilities with the control cells.Results(1)The designed siRNA did efficiently knockdown the expression of NSPc1;(2)Transient knockdown of NSPc1 could repress BrdU incorporation;(3) The established NSPc1-knockdown cell lines had a significantly lower proliferation rate than that of control cells.Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells.The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.