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Objective To construct a lentiviral vector for overexpression of bone morphogenetic protein 7(BMP7)in mice,and the effect of BMP7 overexpression on the expression of Jagged1 in mouse aortic endothelial cells and the calcification of the co-cultured vascular smooth muscle cells(VSMCs)were analyzed.Methods According to the target gene information Mouse-BMP7(NM_007557.3)and plasmid information pLVX-zsGreen-C1,gene sequence synthesis was carried out to construct BMP7 overexpression lentivirus.The efficiency of BMP7 overexpression lentivirus infection was detected by qPCR;the expression of Jagged1 protein in aortic endothelial cells from infected mice was detected by Western blot.The endothelial cells with lentivirus overexpressing BMP7 were co-cultured with VSMCs,and the calcification of VSMCs was observed by alizarin red staining.Results BMP7 overexpression lentiviral vector was successfully constructed and transfected into aortic endothelial cells.qPCR test results showed that the expression level of BMP7 mRNA was significantly increased in the BMP7 overexpression group than that in the normal control group(P<0.01),while there was no significant difference in the expression of BMP7 mRNA between the empty vector control group and the normal control group(P>0.05).Western blot results showed that the expression level of Jagged1 protein in endothelial cells of mouse in the BMP7 overexpression group was significantly lower than that in the normal control group(P<0.01),while there was no significant difference in the expression level of Jagged1 protein in endothelial cells between the empty vector control group and the normal control group(P>0.05).The results of alizarin red staining showed that the calcification of VSMCs was significantly increased after co-cultured with endothelial cells infected with BMP7 lentivirus.Conclusion Mouse BMP7 overexpression lentiviral vector was successfully constructed,and overexpression of BMP7 can reduce the expression of Jagged1 in mouse aortic endothelial cells and promote the calcification of co-cultured VSMCs.
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Objective To construct mouse BaF3-FIP1L1-PDGFRA(F/P),BaF3-F/P-T674I and BAF3-F/P-D842V pre-B cell strains which stably express F/P fusion protein and its T674I and D842V mutants in order to e-valuate their activity by checking their responses to tyrosine kinase inhibitors(TKIs).Methods Lentivirus infected technique was used to transfect the target gene into BaF3 cells.RT-qPCR was used to detect mRNA expression,and CCK-8 method was used to detect the inhibitory effect of TKIs on the proliferation of stable cell strains.Results The constructed BaF3-F/P,BaF3-F/P-T674I and BAF3-F/P-D842V cell strains all transcripted FIP1L1 and PDGFRA mRNA.They exhibited malignant phenotypic characteristics of proliferation independent of IL-3 and sen-sitivity to corresponding TKIs.Conclusions The pre-B-cell strains stably expressing F/P and its T674I and D842V mutants are successfully constructed,which provide a good cell model for the development of compounds targeting at those molecules.
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A 11-year old female patient with severe thalassemia, receipt a lentivirus-based cell and gene therapy (CGT) therapy in Shenzhen Children′s Hosptial on July 27th, 2021. At the two follow-up visits after discharge, patient were continuously tested positive for HIV screening through HIV Ag/Ab Combo assay (chemiluminescence Immunoassay), and the viral load results of HIV-1 nucleic acid testing (NAT) were both>5 000 copies/ml. The patient can be diagnosed with HIV infection according to the National Guideline for Detection of HIV/AIDS(2020 Revised Edition). The thorough investigation findings and supplementary experiment results indicated that the false-positive HIV-1 NAT results was caused by cross-reactivity between the target sites detected by conventional HIV-1 NAT reagents and the lentiviral vectors fragments integrated into the genome of patient′s hematopoietic stem/progenitor cells. In conclusion, it is important for laboratories to select appropriate HIV-1 NAT testing platforms which won′t cause cross-reactivity for the testing of samples from patients who have been treated with HIV-derived vectors. It is also recommended to design and develop NAT testing platforms with multiple target regions labeled by different fluorescents for HIV NAT supplementation experiment to reduce the risk of false-positive diagnoses of HIV infection.
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Objective @#To study the effect of sex-determining region Y-frame protein 3 (SOX3) on proliferation and estradiol secretion in human ovarian granulosa cells (KGN cell line) . @*Methods@#The gene sequence of human SOX3 (NM_005634. 3) was searched in Gene-Bank , an NCBI database , and the target gene SOX3 was amplified by PCR , which was cloned into lentiviral vector pLV-EF1a-GFP-2A-Puro to obtain the overexpression lentiviral re- combinant plasmid pLV-EF1a-GFP-2A-Puro- SOX3 ; the correctly sequenced overexpressed lentiviral recombinant plasmid as well as packaging plasmids ( pGag/Pol , pRev , pVSV-G) were co-transfected into human embryonic kidney cell line ( HEK 293T) cells ( pLV-SOX3 group) , and pLV-EF1a-GFP-2A-Puro and packaging plasmids (pGag/Pol , pRev , pVSV- G) were co-transfected into HEK 293T cells (pLV-NC group) , the lentiviral particles of both groups were collected and the titers of the viruses were measured after 48 h of transfection , the lentiviruses of the two groups were infected into KGN cells , and the stably expressed cell lines were obtained after puromycin screening for 2 weeks; real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the SOX3 mRNA and protein levels in the two groups; CCK-8 assay was used to detect the proliferative ability of the cells in the two groups; ELISA was used to determine the concentration of estradiol in the two groups .@*Results@#The identification of PCR products and sequencing results showed that the SOX3 gene fragment was amplified successfully , and the enzyme digestion and sequencing results indicated that the construction of overexpression lentiviral recombinant plasmid was completed; green fluorescence could be detected after lentiviral infection of HEK 293T cells , which indicated that lentiviral packaging was successful; the lentivirus was screened by puromycin after lentiviral infection of KGN cells , and the cells were observed to express green fluorescence under the fluorescence microscope; RT- qPCR and Western blot assays both showed that the expression level of SOX3 in the pLV-SOX3 group was significantly higher than that in the pLV-NC group ( P < 0. 05) . CCK-8 assay results showed that the proliferation ability of the cells in the pLV-SOX3 group significantly increased compared with that in the pLV-NC group (P < 0. 01) . ELISA results showed that estradiol concentration was elevated in the pLV-SOX3 group com- pared with the pLV-NC group (P < 0. 05) . @*Conclusion@#Overexpression of the transcription factor SOX3 can pro- mote the proliferation and estradiol secretion of human ovarian granulosa cells KGN .
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Objective @#To construct lentiviral vector of p62 gene over expression , and stably express p62 gene in human monocytic leukemia cells 1 (THP 1) , and to provide a way to study the role of p62 gene at the cellular lev el .@*Methods @#The p62 gene fragment was amplified by polymerase chain reaction (PCR) , and the amplified product was ligated to the linearized pcDNA3 1 Flag PCDH10 lentiviral vector. After identifying with PCR , the PCR product was cotransfected with the packaging plasmid into human embryonic kidney cells 293 (HEK 293T) . THP 1 cells were infected with recombinant lentivirus . Positive cell clones were screened by ampicillin . Western blot and real time fluorescence quantitative polymerase chain reaction (RT qPCR) were used to detect THP 1 cell lines with high p62 expression ( overexpression group) and THP 1 cell lines transfected with empty plasmid without p62 gene ( control group) . The expression levels of TNF α, IL 1βand Cxcl1 after K. p. infection were detected by RT qPCR .@*Results @#The p62 gene fragment was successfully obtained by PCR and ligated to pcDNA3 1 Flag PCDH10 vector. PCR confirmed that p62 pcDNA3 1 Flag PCDH10 recombinant plasmid was constructed successfully. Am picillin resistant cell lines were selected after lentiviral infection of THP 1 cells . The results of Western blot analysis showed that the THP 1 cells with drug sieve survival increased the expression of P62 protein compared with the con trol cells (P < 0.001) , and RT qPCR analysis showed that the relative mRNA expression of p62 increased (P < 0.001) . THP 1 cells with high expression of P62 were successfully constructed . The levels of TNF-α、IL-1βand C xcl1 from THP 1 cells with high expression of P62 significantly increased after infection with K. p. (P < 0.01) . @*Conclusion @#P62 pcDNA3 1 Flag PCDH10 vector and THP 1 cells with high expression of P62 can be successful ly constructed by three plasmid packaging system , which provides a basis for the study of p62 .
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Objective To establish the human colorectal cancer cell line HCT116 with ZNF24 gene overexpression by constructing lentiviral vector of ZNF24 gene overexpression, and provide material basis for subsequent research. Methods The recombinant expression ZNF24 lentiviral plasmid pMT-ZNF24 was constructed by the homologous recombination of ZNF24 gene and 3FLAG tag sequence fragments which was amplified by PCR into lentiviral vector pMT-406.The recombinant plasmid pMT-ZNF24 and the auxiliary packaging vector plasmids pCMV-dR8.9 and pCMV -VSV-G were co-transfected into 293T cells, after which the lentivirus were collected. The virus titer was determined with well dilution method. The lentivirus was transfected into HCT116 cells, and the expression levels of ZNF24 were detected by qRT-PCR and Western blot. Results The recombinant vector pMT-ZNF24 was successfully constructed, and the corresponding virus was obtained. The virus titer was 3.25×109 TU/ml. The expression levels of ZNF24 in cells transfected with recombinant ZNF24 lentivirus were significantly higher than those in blank and negative control. Conclusion The ZNF24 gene overexpression lentiviral vector had been constructed , and the corresponding virus and the HCT116 cell line expressing ZNF24 had been obtained .
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Objective:To evaluate the feasibility of 8E5 cells and CD19-CAR-Jurkat cells used as reference cells in the detection of lentiviral vector integration sites with different methods.Methods:Single clones of 8E5 cells and CD19-CAR-Jurkat cells were selected using limiting dilution method. Digital PCR was established to detect the copy number of HIV-1 in 8E5 cells and the copy number of CAR in CD19-CAR-Jurkat cells. High-throughput sequencing techniques (whole-genome resequencing, modified genome sequencing and probe hybridization capture) were used to detect integration sites in 8E5 cells and CD19-CAR-Jurkat cells, and optical genome mapping (OGM) technology was used for further confirmation.Results:Three clones of 8E5-D8 cells and six clones of CD19-CAR-Jurkat 2-6 cells were selected using the limiting dilution method. 8E5-D8 and CD19-CAR-Jurkat 2-6 were chosen as candidate cells based on their gene copy numbers detected by digital PCR and flow cytometry. These cells were then expanded and cryopreserved. Digital PCR showed that 8E5-D8 cells contained approximately 1 copy per cell, while CD19-CAR-Jurkat 2-6 cells contained approximately 13 copies per cell. High-throughput sequencing revealed one integration site in 8E5 cells and 13 integration sites in CD19-CAR-Jurkat cells, which matched the copy number detection results. All these integration sites were further confirmed at the submicroscopic level of chromosomes using OGM.Conclusions:Based on the insertion copy numbers and integration sites, 8E5-D8 cells and CD19-CAR-Jurkat 2-6 cells could be used as reference cells in further development of methods for detecting integration sites in CAR-T cell lentiviral vectors.
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As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)
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Animales , Bovinos , Cigoto , Animales Modificados Genéticamente/genética , Transgenes , Embrión de Mamíferos , Vectores Genéticos/análisis , Fertilización In Vitro/veterinaria , Técnicas de Transferencia de Gen/veterinariaRESUMEN
OBJECTIVE: To construct the RNA interference(RNAi) lentiviral vector of suppressor of variegation 3-9 homolog 1(Suv39 h1) and verify its interfering efficiency by transfecting it to the bone marrow-derived mesenchymal stem cells(BMSCs). METHODS: The oligonucleotides of RNA plasmid were designed and synthesized according to the gene sequence of Suv39 h1 and short hairpin RNA design principles. Three kinds of LV-Suv39 h1-RNAi recombinant plasmids with different lentivirus knockdown targets(KD1, KD2 and KD3) were constructed. After identification by restriction analysis and sequencing, the packaged lentivirus vectors with the three kinds of Suv39 h1 gene were transfected into rat BMSCs at logarithmic growth stage, and were named KD1, KD2 and KD3 transfection groups. The control group was transfected with the negative control virus. After 72 hours transfection, the transfection efficiency was evaluated, and the relative mRNA levels of Suv39 h1 were determined by quantitative real-time polymerase chain reaction(qPCR). RESULTS: Sequencing analysis demonstrated that three kinds of LV-Suv39 h1-RNAi recombinant plasmids were constructed correctly. The results of transfection efficiency evaluation showed that more than 80.00% green fluorescence was expressed in the BMSCs transfected with the three lentiviral vectors with a multiplicity of infection of 20. These results indicated that lentivirus was successfully constructed and transfection efficiency was high. The results of qPCR showed that the relative expression of Suv39 h1 mRNA in BMSCs of KD1, KD2 and KD3 transfection groups was lower than that in the control group(all P<0.05), and the relative expression of Suv39 h1 mRNA in KD1 and KD3 transfection groups was lower than that in KD2 transfection group(both P<0.05). However, there was no significant difference in the relative expression of Suv39 h1 mRNA between KD1 and KD3 transfection groups(P>0.05). CONCLUSION: The constructed lentiviral vector with low expression of Suv39 h1 was constructed successfully. This vector can be expressed in rat BMSCs, which lays a foundation to study the effect of Suv39 h1 gene in acute myeloid leukemia.
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ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.
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Humanos , Técnicas In Vitro , Inmunoterapia Adoptiva , Antígenos CD19 , Citotoxicidad Inmunológica , XenoinjertosRESUMEN
Objective: In this study, we investigated the antitumor activity of interleukin (IL)-18 on A549 human lung cancer cell line and evaluated the potential of IL-18 therapy in lung cancer. Materials and Methods: We generated a human IL-18 lentiviral expression vector and examined three groups of A549 cells, including nontransduced cells and cells transduced with either IL-18 or an empty lentiviral expression vector. IL-18 expression, cell proliferation, and apoptosis were examined using Western blotting, methylthiazolyldiphenyl-tetrazolium bromide assay, and flow cytometry, respectively. The expression of the T helper 1 (Th1) cytokine interferon-γ (IFN-γ) and Th2 cytokine IL-4 was analyzed by enzyme-linked immunosorbent assay (ELISA). Results: Compared to the other groups of cells, A549 cells transduced with the IL-18 lentiviral expression vector exhibited significant increases in IL-18 expression, apoptosis, and the fraction of cells in G0 and G1 phases of the cell cycle and significant decrease in proliferation. Furthermore, ELISA results showed that IFN-γ expression increased significantly and IL-4 decreased in A549 cells transfected with IL-18 lentivirus expression vector. Conclusion: Using a lentiviral expression vector, IL-18 was expressed stably and efficiently in A549 cells, which showed attenuated proliferation and tumor cell growth, and enhanced tumor cell apoptosis. IL-18 expression also induced the secretion of IFN-γ, while decreasing the production of IL-4, therefore restoring the balance between Th1/Th2 cell subsets. These findings further demonstrated the antitumor activity of IL-18 and might open new therapeutic avenues for the prevention and treatment of lung cancer.
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BACKGROUND: Lentiviral vectors have been widely used as exogenous transgenic vectors. However, a recombinant lentiviral vector containing rat diacylglycerol kinase y (DGKy) gene has not been reported. OBJECTIVE: To construct lentiviral overexpression vector of rat DGKy by homologous recombination. METHODS: Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats, and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKy gene CDS. Then, the two homologous recombination fragments were ligated into the plasmid vector. The positive clones were confirmed by PCR and DNA sequencing. The CMV-rat DGKy-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells. The expression of GFP in infected 293T cells was observed under fluorescence microscope. Real-time PCR and western blot assay were used to detect the relative expression of DGKy mRNA in infected 293T cells. RESULTS AND CONCLUSION: The results of PCR simplification and sequencing indicated that the CMV-rat DGKy-GFP lentiviral vector was successfully constructed. In 293T cells infected with CMV-rat DGKy-GFP lentivirus, the expression of GFP was observed under fluorescence microscope and the DGKy mRNA expression was increased significantly than that of the vector group by real-time PCR (P < 0.01). Western blot assay results showed that the DGKy protein expression of the selected GFP-positive 293T cells was increased very significantly (P < 0.001). To conclude, the rat DGKy lentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells.
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BACKGROUND: Connexin 43 (Cx43) plays an important role in occurrence and development of osteoarthritis. However, the specific mechanisms involved remain unclear. OBJECTIVE: To verify the possibility of the dominant position of Cx43 in connexin family in osteoarthritis by detecting the expression of Cx43 in articular cartilage and chondrocyte cell line, and to construct shRNA lentivirus vector of Cx43 gene and establish a stable transfer cell line of chondrocyte (SW1353). METHODS: Animal models of osteoarthritis were established in six C57BL/6 mice by anterior cruciate ligament transection. The differences of Cx43 expression between osteoarthritic and normal knees were investigated by immunohistochemistry. Expression of Cx43 mRNA in chondrocyte (SW1353) was detected by RT-PCR, and the expression levels of Cx37, Cx40, Cx45 and Cx46 in SW1353 cells were detected as control. Cx43 were connected to the lentiviral vector carrying the EGFP gene, to reconstruct the lentiviral vector plasmid. The viral particles were generated by co-transfection of 293T cells with Cx43-shRNA. After transfection of Cx43-shRNA lentiviral vector into chondrocytes (SW1353), the expression level of Cx43 was detected by western blot assay and RT-PCR. The study protocol was approved by the Ethics Committee of State Key Laboratory of Oral Diseases, approved No. SKLODLL2013A172. RESULTS AND CONCLUSION: The expression level of Cx43 was significantly increased in the articular cartilage of osteoarthritic knees. The expression level of Cx43 mRNA was significantly higher than that of Cx37, Cx40, Cx45 and Cx46 in chondrocytes (SW1353). In SW1353 cells, Cx43 occupied the dominant position in connexin family. Cx43 shRNA lentiviral vector could inhibit the expression of Cx43 mRNA in SW1353 cells. The stably transfected SW1353 cell line was screened, laying a foundation for verifying the role of Cx43 in osteoarthritis.
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By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.
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Humanos , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Genética , Lentivirus , Genética , MicroARNs , Metabolismo , Receptor de Muerte Celular Programada 1 , Regiones Promotoras Genéticas , GenéticaRESUMEN
Alzheimer's disease (AD) and Parkinson's disease (PD) are common neurodegenerative diseases in human. The pathogenesis of AD and PD is complex, and the current drugs and surgical treatments have not successfully alleviated or terminated the progression of the diseases. The lentiviral vector (LV) is a retroviral vector. In recent years, LV mediated gene therapy has been a hotspot to study the mechanisms of human disease and clinical drug discovery. This review summarizes the recent progresses in the treatment of AD and PD by the application of LV, and offers a prospect for its application.
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Humanos , Enfermedad de Alzheimer/terapia , Terapia Genética , Enfermedad de Parkinson/terapiaRESUMEN
@#【Objective】Using the CRISPR/Cas9（CRISPR/crispr-associated（Cas）9 method，a dual-target lentiviral vector containing single- guide RNAs（sgRNAs）targeting both the 5’and 3’ends of the anti- differentiation noncoding RNA（DANCR）gene was constructed. Stable knockout of DANCR gene in mesenchymal stem cells（MSC）would be helpful for the future study of the biological function of DANCR.【Methods】Designed sgRNAs targeting either the 5’or 3’ end of DANCR and cloned into two CRISPR vectors. The vector was transfected into 293FT cells，and the genomic DNA of 293FT cells was extracted to verify the efficiency of individual sequence. Two functional sgRNAs targeting either the 5’ or 3’end were incorporated into a same lentiviral CRISPR vector through gateway and enzymatic ligation. 293FT was used for lentiviral packaging，after which the virus was harvested to infect MSC，and the knockout efficiency of DANCR in MSC was detected.【Results】All four sgRNA sequences targeting DANCR successfully guided Cas9 to cleave the gene. sgRNAs targeting either the 5’and 3’end were combined to establish a dual-target lentiviral vector for stable knockout of DANCR. The vector was packaged into lentivirus and infected MSC. Finally，we successfully obtained mesenchymal stem cell lines with DANCR gene knockout.【Conclusions】Using the CRISPR method，a dual-target lentiviral vector can efficiently and stably knock out DANCR gene in MSC.
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Objective: To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and to explore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods: The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoR I and Age I restriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results: The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU · mL-1 and 5 × 108 TU · mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12. 640 0 ± 0. 788 4) was significantly increased by 15. 07 times (t=14. 72, P<0. 01) compared with control group (0. 838 7 ± 0. 145 6). Conclusion: The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus successfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.
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Objective: To construct a lentiviral vector with overexpression of endoplasmic reticulum protein 29 ERp29), and to explore the effects of ERp29 overexpression on the proliferation, migration and invasion of gastric cancer MGC803 and SGC7901 cells. Methods: The lentiviral plasmid with ERp29 pCDH-ERp29) tagged with red fluorescentce protein and control plasmid pCDH-Vector) were constructed, and then the MGC803 and SGC9901 cells were infected with these lentivital vectors. Under fluorescence microscope, the infection of these cells were observed. CCK-8 assay and clone formation assay were performed to test the proliferation abilities of MGC803 and SGC7901 cells infected with ERp29. Transwell chamber assay was used to test the abilities of migration and invasion of MGC803 and SGC7901 cells infected with ERp29. Real-time PCR and Western blotting methods were used to detect the expression levels of ERp29 mRNA and protein in MGC803 and SGC7901 cells which were stably infected by ERp29, respectively. Results: The enzyme digestion identification result showed that the pCDH-ER29 overexpression vector was successfully constructed. The fluorescence microscope result showed the successful infection in the gastric cancer cells. The CCK-8 assay and clone formation assay results showed that compared with pCDH-Vector group, the proliferation ability of the cells in pCDH-ERp29 group had no marked changes (P > 0.05). Transwell chamber assay revealed that compared with pCDH-Vector group, the number of migration and invasion cells in the MGC803 and SGC7901 cells in pCDH-ERp29 group were significantly decreased (P < 0 . 05). The expression levels of ERp29 mRNA and protein in the cells in pCDH-ERp29 group were significantly increased compared with pCDH-Vector group (P < 0 . 05). Conclusion: The lentiviral vector with overexpression of ERp29 is constructed successfully, and the overexpression of ERp29 in MGC803 and SGC7901 cells can significantly inhibit their abilities of migration and invasion.
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Objective: To isolate, culture and identify rabbit bone mesenchymal stem cells (BMSCs) so as to explore the optimal conditions for lentiviral vector-mediated enhanced green fluorescent protein (eGFP) infection in rabbit BMSCs and screen stable transfected BMSCs in rabbits. Methods: BMSCs were obtained by whole bone marrow adherence method. The osteogenic, chondrogenic and adipogenic differentiation of BMSCs was made by alizarin red, toluidine blue and oil red O staining, respectively. The expressions of CD44 and CD90 were detected by immunofluorescence. The concentration of puromycin was used to screen the minimum lethal concentration of BMSCs; the lentiviral vector with multiplicity of infection (MOI) of 50, 100, 150 and 200 mediated eGFP BMSCs were infected; the fluorescence expression was observed under an inverted microscope, and the stable transformation system was screened with puromycin. Results: When MOI was 150, lentiviral vector-mediated eGFP infection of rabbit BMSCs was the most efficient. The optimum concentration of puromycin for stable transfection of rabbit BMSCs was 1.0 μg/mL. Conclusion: Rabbit BMSCs were successfully cultured in this experiment. The stem cells were labeled with lentivirus-mediated GFP and stable transfected rabbit BMSCs were screened. A simple and effective stem cell labeling method was established to label BMSCs in vivo.
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OBJECTIVE@#To construct a recombinant lentiviral expression vector pCDH-Daxx-EGFP to investigate the effect of Daxx on the proliferation of vascular smooth muscle cells (VSMCs).@*METHODS@#The recombinant lentiviral expression vector pCDHDaxx-EGFP was constructed using PCR-based accurate synthesis method. After identification by sequencing and enzyme digestion, the recombinant lentiviral vector was contransfected into 293T cells with lentivirus packaging vector. The recombinant lentivirus particles were collected and purified to infect VSMCs, whose expression of Daxx was detected with Western boltting. The cells infected with the empty vector pCDH-EGFP or pCDH-Daxx-EGFP were incubated in serum-free medium or in the presence of angiotensin Ⅱ (AngⅡ). The cell viability was determined with MTT assay, and the cell cycle changes were analyzed with flow cytometry. The cell migration ability was assessed using a scratch wound healing assay. The expression of p-Akt protein in the cells was detected using Western blotting.@*RESULTS@#Double enzyme digestion and sequencing confirmed successful construction of the recombinant plasmid. Compared with the cells infected with the empty vector, the cells infected with pCDH-Daxx-EGFP exhibited significantly increased expressions of Daxx protein ( < 0.05). AngⅡ treatment of the cells infected with the pCDH-Daxx-EGFP, as compared with the cells infected with the empty vector, significantly lowered the cell viability, S phase cell ratio and cell migration ability ( < 0.05), and significantly decreased the expression level of p-Akt protein ( < 0.05).@*CONCLUSIONS@#We successfully constructed the recombinant lentiviral vector pCDH-Daxx-EGFP and overexpressed Daxx in primary cultured VSMCs using this vector. Daxx overexpression can inhibit AngⅡ-induced proliferation and migration in VSMCs probably by regulating p-Akt protein.