Small interfering RNA (shRNA)(target sequence: 5'-TCACCGTGAAGATCATGAA-3') targeting human SYT7 was designed and the scrambled shRNA(target sequence: 5'-TTCTCCGAACGTGTCACGT-3') was used as negative control. Lentivirus expressing such SYT7 shRNA and scrambled shRNA were constructed (GeneChem, Shanghai, China). Transfection was performed in RKO cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY) as the manufacturer's protocol. After 72 hours, the transfection efficiency could be detected using fluorescence microscope. Then cells were harvested and total RNA and protein were extracted to determine the knockdown efficiency using real-time PCR and western blot. Finally, LV transfected RKO cells were quickly collected for later experiment.
Lentivirus expressing
Manufactured by Genechem
Sourced in China
Lentivirus expressing is a laboratory equipment used for the production and delivery of lentiviral vectors. Lentiviral vectors are a type of virus-based system used to efficiently introduce genetic material into target cells, including non-dividing cells. The core function of this equipment is to facilitate the generation and handling of lentiviral particles for various research and applications.
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Lab products found in correlation
5 protocols using lentivirus expressing
1
Lentiviral shRNA-Mediated Knockdown of SYT7
2
Tracking Transplanted ADMSCs via EGFP Labeling
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In order to track the transplanted ADMSCs in vivo, third-passage ADMSCs were plated at a density of 1 × 106 cells per flask (T25) and then transduced with lentivirus expressing enhanced green fluorescence protein (EGFP) (Shanghai Genechem Co. Ltd., Shanghai, China) 3 days prior to transplantation. The optimal multiplication of infection (MOI) in transduction medium containing 8 mg/L polybrene was 20. The medium was replaced 24 h after transduction. The efficiency of transduction was assessed by monitoring EGFP gene expression by fluorescence microscopy. Cell viability was determined prior to transplantation by trypan blue staining. Labeled cells were used for transplantation at a concentration of 1 × 105 cells/µL.
3
Overexpression and Knockdown of GASP1
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Lentivirus expressing GASP1 and control lentivirus were purchased from Shanghai Genechem Co., Ltd. One day before infection, cells were plated and allowed to grow to 30–50% confluence. Positive cells were selected by puromycin and kept in the solution containing a low concentration of puromycin for the subsequent experiments. The efficiency of overexpression was confirmed by qRT-PCR and western blot assays.
Lentivirus expressing Cas9 and sgRNAs targeting GASP1 and control lentivirus were obtained from HanBio Tech (Shanghai, China). Cells were infected with the above lentiviruses (MOI of 20) and selected with puromycin for 7 days. GASP1 expression was then determined by western blot assays. The sgRNA sequences used in this study were presented in Supplementary Table2 .
Lentivirus expressing Cas9 and sgRNAs targeting GASP1 and control lentivirus were obtained from HanBio Tech (Shanghai, China). Cells were infected with the above lentiviruses (MOI of 20) and selected with puromycin for 7 days. GASP1 expression was then determined by western blot assays. The sgRNA sequences used in this study were presented in Supplementary Table
4
Endothelial Cell Culture and Lentiviral Transduction
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Primary HUVECs, human EECs, human CAECs, human MCECs, and human AECs were obtained from Clonetics Inc. (Walkersville, MD, USA). Cells were grown in endothelial basal medium supplemented with 2% fetal bovine serum and penicillin (100 u/ml), and streptomycin (100 µg/ml). Cultured cells were used between passages 3 and 8. All cells were incubated in a humidified atmosphere of 5%CO2 + 95% air at 37 °C. When 70–80% confluent, the cells were treated with different agents. For HEK293 cells, cells were cultured in M200 medium supplemented with 2% fetal bovine serum and penicillin (100 u/ml), and streptomycin (100 µg/ml). For lentivirus infection, cells were infected with lentivirus expressing AP-2α shRNA or circRNA-RBCK1 shRNA from Shanghai Genechem Co., Ltd. (Shanghai, China) overnight in antibiotics-free medium supplemented with 2% FBS. The target sequence of CircRNA-RBCK1 shRNA is TCTTGCAGCAGTGGGTGATTG. The target sequence of AP-2α shRNA is TCCCAGATCAAACTGTAATTA. These targets were designed by VectorBuilder Inc. The cells were then washed and incubated in fresh medium for an additional 12 h before experiments.
5
Lentiviral-Mediated Knockdown and Overexpression of IRF4
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For IRF4 knockdown, the lentivirus expressing short hairpin RNA (shRNA) targeting the sequence of IRF4 (5′-GCCGTACAAAGTTCAGGATCC-3′) and the control sequence (5′-TTCTCCGAACGTGTCACGT-3′) was purchased from Genechem Co. Ltd (Shanghai, China). lentivirus expressing scrambled shRNA was used as a negative control (sh-Ctrl; Genechem Co. Ltd.). For IRF4 overexpression, the targeted IRF4 gene was amplified in human sample by PCR with the following primers: forward: 5′-CACCATGACAACGCCTTACC-3′ and reverse 5′-CATTTTCACAAGCTGGGCCT-3′. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector to construct a lentiviral vector carrying IRF4. The empty lentivirus vector was used as a negative control (Ctrl). The shRNA-expressing, overexpressing lentivirus and the corresponding controls were transfected into 293 T cells, which are always used in producing lentiviruses (19 (link)), together with the lentivirus helper plasmids to generate respective lentivirus. After transfection, the lentivirus supernatant was collected to transfect A549 and LC-AI cells. Infectious lentivirus was harvested 48 h post-transfection, centrifuged to remove cell debris, and then filtered through 0.45 µm cellulose acetate filters.
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