Construction and expression of lentiviral vector overexpressing ZNF24 gene in human colorectal cancer HCT116 cell / 药学实践杂志

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 .

Construction and identification of a HEK293 cell line with stable TrxR1 overexpression / 南方医科大学学报

OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

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)

Advances in quantification of lentiviral vectors / 生物工程学报

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.

Construction and verification of RNA interfering lentiviral vector targeting Suv39h1 gene / 中国职业医学

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.

Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies

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.

Antitumor activity of interleukin-18 on A549 human lung cancer cell line

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.

Progress in lentiviral vector-mediated gene therapy for Alzheimer's disease and Parkinson's disease / 生物工程学报

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.

Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299 / 解剖学报

To investigate the proliferation and apoptosis of lung adenocarcinoma cells line HI299 by the lentiviral vector mediated RNA binding protein quaking-5 ( QKI-5). Methods GV358 ( up-regulation ) and GV248 ( down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of HI299 cells was determined by the detection of the expression membrane protein V (annexin V ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of HI299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of HI299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.

Construction of a novel lentiviral vector knocking down PD-1 via microRNA and its application in CAR-T cells / 生物工程学报

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.

Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination / 中国组织工程研究

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.

Expression of connexin 43 in cartilage and chondrocyte of osteoarthritis and construction of shrna lentiviral vector targeting connexin 43 / 中国组织工程研究

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.

Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method / La eficiencia de la expresión transg é nica en células bovinas varía según el tipo de célula y el método de la transferencia de genes / A eficiência da expressão do transgene em células bovinas varia de acordo com o tipo celular e o método de transferência gênica

Abstract Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.

Resumen Antecedentes: La producción de animales transgénicos sigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.

Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde - GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.

Stable Knockout of lncRNA DANCR Gene in Mesenchymal Stem Cells with a Dual-Target CRISPR Lentiviral Vector / 中山大学学报(医学科学版)

@#【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.

Lentiviral vector mediated enhanced green fluorescent protein screening for stably transfected rabbit bone marrow mesenchymal stem cells / 西安交通大学学报(医学版)

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.

Construction of lentiviral vector with overexpression of ERp29 and its effect on biological behavior of gastric cancer cells / 吉林大学学报(医学版)

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.

Construction and identification of miR-186 overexpression lentiviral vector / 吉林大学学报(医学版)

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.

Daxx overexpression inhibits AngⅡ-induced proliferation and migration in vascular smooth muscle cells / 南方医科大学学报

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.

Construction and identification of recombinant lentiviral plasmid of receptor for advanced glycation end products / 国际生物医学工程杂志

Objective To construct a recombinant lentiviral plasmid of receptor for advanced glycation end products (RAGE) and establish RAGE stable expressing hepatocellular carcinoma cell models in order to explore the pathogenesis of RAGE in the occurrence and development of hepatocellular carcinoma. Methods RAGE gene fragments were amplified by PCR and cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro. Then the recombinant lentiviral plasmid and packaging plasmid were used to package lentivirus in HEK293T cells by calcium phosphate transfection. Lentivirus supernatant was collected to infect hepatoma cells HepG2 and Huh7. After that cells were screened by puromycin. Finally, RAGE expression was detected by real-time PCR and Western blot. Results RAGE gene fragment was successfully amplified and inserted into lentiviral expression vector. After packaging the lentivirus-infected liver cancer cells, real-time quantitative PCR and Western blot results showed that the mRNA (P<0.05) and protein expression of RAGE cells stably expressing RAGE were significantly higher than that of the control cells. Conclusions The RAGE overexpressing hepatocarcinoma cell line was successfully constructed by using lentiviral expression vector, which laid a good foundation for further study on the pathogenesis of RAGE.