BC cell lines, including MDA-MB-231 (SCSP-5043), MCF-7 (SCSP-531), MDA-MB-468 (TCHu136), and BT549 (TCHu 93), non-tumorigenic breast epithelial cell line MCF-10A (SCSP-575), and HEK293T cells (SCSP-502) were obtained from the Chinese Academy of Sciences (Shanghai, China). The conditions for cell culture were described in a previous study [21 (link)]. All siRNAs and their negative control (si-NC) were synthesized by IBSbio (Shanghai, China) and the sequences were listed in Additional file 1 . The plasmids for GFP-fused wild-type (WT), truncated DHX15 and site-mutated DHX15 [P327E, T421A, N422K, Y485E (GFP-DHX15-MUT)] were purchased from IBSbio (Shanghai, China). SiRNAs and plasmids were transfected by Lipo8000 reagent (Beyotime, Shanghai, China). The lentiviral vector pLV-circRNA-Hygro (HarO Life, Shanghai, China) and pCDH-MSCV-MCS-EF1-GFP-puro (IBSbio, Shanghai, China) were applied to construct overexpression plasmid of circRNF10 and DHX15, respectively. After lentiviral packaging using GMeasy Lentiviral Packaging Kit (Genomeditech, Shanghai, China) and cell infection, the cells were selected by 1 μg/mL puromycin (Beyotime, Shanghai, China) or 100 μg/mL hygromycin B (HarO Life, Shanghai, China), according to the antibiotic resistance.
Gmeasy lentiviral packaging kit
Manufactured by Genomed
Sourced in China
The GMeasy Lentiviral Packaging Kit is a laboratory tool designed for the production of lentiviral particles. It contains the necessary components to generate lentiviral particles for various research applications.
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Lab products found in correlation
4 protocols using gmeasy lentiviral packaging kit
1
Engineered Cell Lines for Research
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Lentiviral Transduction of HEK293T Cells
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Lentiviral particles were prepared using the GM easy™ lentiviral packaging kit (Genomeditech, Shanghai, China). In brief, HEK293T cells were seeded at a density of 2 × 106 in a 10-cm cell culture dish containing 10 ml of media. The next day, the cells were co-transfected with a complex containing the recombinant lentiviral construct expressing NS5A or EGFP (pTRIP-NS5A or pTRIP-EGFP), and three helper plasmids (GAG/Pol, REV and VSV-G from the kit) according to the manufacturer’s instructions. The medium was changed 18–20 h after transfection. The supernatant was collected after an additional 48 h. The viral particles were concentrated and stored at − 80 °C until use. The titer of the viral particles was determined by quantitative real-time PCR (the primers are listed in Table 1 ).
Primers used to detect gene expressions at mRNA level
| Gene name | Primer sequences (5′-3′) |
|---|---|
| NS5A | F: 5′-ATTGGCTGCGTGACATCTG-3’ |
| R: 5′-ACCACGCTCTGCTCCTCACT-3’ | |
| Mouse SREBP-1c | F: 5′-GGAGCCATGGATTGCACATT-3’ |
| R: 5′-GCTTCCAGAGAGGAGCCCAG-3’ | |
| Mouse FASN | F: 5′-CACAGATGATGACAGGAGATGGA-3’ |
| R: 5′-TCGGAGTGAGGCTGGGTTGATA-3’ | |
| Mouse ACC1 | F: 5′-TGTTGGGGTTATTTCAGTGTTGC-3’ |
| R: 5′-TGTCCAGCCAGCCAGTGTCG-3’ | |
| Mouse β-Actin | F: 5′-TTCCTTCTTGGGTATGGAAT-3’ |
| R: 5′-GAGCAATGATCTTGATCTTC-3’ |
F Forward primer, R Reverse primer
3
Lentiviral-mediated TREM2 knockdown in GC cells
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The small hairpin RNA (shRNA) oligonucleotides against human TREM2 (target region 1, 5′-GAGCCTCTTGGAAGGAGAAAT-3′; target region 2, 5′-CACAGCCATCACAGACGATAC -3′) and control shRNA non-specific control oligonucleotides were annealed and inserted into the BamH I-EcoR I site of pPLK/GFP+puro vector. Viral packaging was implemented in HEK293T cells using the GM easyTM Lentiviral Packaging Kit (Genomeditech, Shanghai). The supernatant conditioned viral particles were harvested at 48 and 72 h after transfection and the filtered through 0.45 μm filters. After viral infection, GC cells were incubated with filtered conditioned media for 8 h. Puromycin was then added to select lentivirus-infected cells. After 2 weeks, experiments were performed.
4
Lentivirus-mediated miR-26a Delivery
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The lentivirus expressing miR-26a (LV3-miR-26a) was generated using a GM EasyTM Lentiviral Packaging Kit (Genomeditech, China) according to the manufacturer’s instructions. Briefly, the pGMLV vector and GM EasyTM Lentiviral Mix plasmid were co-transfected with the pCDH1-expressing vector containing the miR-26a sequence into HEK293 cells using HG TransgeneTM Reagent (Genomeditech, China). The lentiviruses were propagated in HEK293 cells and purified using centrifugation and filtration by 0.45 μm Millipore filter membranes. Titering was assayed using a serial dilution method. For local delivery of the lentiviral vector-mediated miR-26a mimics (LV3-26a) and negative controls (LV3-NC) into grafted veins, we used an established local delivery model with Pluronic F-127 gel (Sigma) as previously described58 (link). Vein segments were immersed in 500 μl lentivirus solution (either LV3-miR-26a or LV3-NC, 108 pfu/ml) for 5 min. Then, 250 μl lentivirus solution was preloaded into 250 μl Pluronic F-127 gel (30% wt/vol) at 4 °C and gently painted around the grafted vein segments. Rats were randomly divided into 3 groups: the LV3-miR-26a-treated group, LV3-NC group and the Sham group. The vein grafts were harvested at week 4 after surgery.
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