Overexpression of miR‐194 and miR‐215 in tumor organoids was conducted using lentiviral vectors. A puromycin‐resistant lentiviral vector (pLVSIN‐CMV Pur) was obtained from (Takara Bio, Shiga, Japan). The sequences of miR‐194, miR‐215 and the cluster were prepared by PCR. The primers employed are listed in Table S1 . To prepare the infection medium, the lentiviral vector encoding miR‐194, miR‐215, the cluster including both miR‐194 and miR‐215, and the empty control vector were transfected into 293FT cells together with ViraPower lentiviral packaging mix (Thermo Fisher Scientific). We incubated the organoids with trypsin for 10 min at 37°C, and then incubated organoid fragments with a small volume of infection medium for 2 h at 37°C. After centrifugation, we discarded the supernatant and resuspended the pellet in Matrigel. We added the culture medium to the infection medium. Twenty‐four hours after transduction, the infected organoids were selected with puromycin (1 μg/mL).
Plvsin cmv pur
Manufactured by Takara Bio
Sourced in Japan
The PLVSIN-CMV Pur is a lentiviral vector system designed for the delivery and expression of genes of interest in target cells. It contains the necessary components for the production of recombinant lentiviral particles, including the viral packaging and envelope proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lentiviral high titer packaging mix, by Takara Bio (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Virapower lentiviral packaging mix, by Thermo Fisher Scientific (1 mentions)
Mscgm bulletkit, by Lonza (1 mentions)
Plvsin cmv pur vector, by Takara Bio (1 mentions)
Dpsc bulletkit, by Lonza (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Csii cmv mcs ires2 bsd, by RIKEN BioResource Center (1 mentions)
Lentiviral high titer packaging mix with plvsin series, by Takara Bio (1 mentions)
Idt codon optimization tool, by Integrated DNA Technologies (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Transit 293 transfection reagent, by Mirus Bio (1 mentions)
6 protocols using plvsin cmv pur
1
Lentiviral Overexpression of miR-194 and miR-215
2
Generation of DPSC Overexpressing PV-MXRA5-FLAG
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Human DPSCs and MSCs were purchased from Lonza Inc. (Walkersville, MD). DPSCs and MSCs were expanded in a specified medium (DPSC: #PT-3005, DPSC BulletKit, Lonza Inc., MSC: #PT-3001, MSCGM BulletKit, Lonza Inc.) and then maintained in low glucose Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Carlsbad, CA) supplemented with 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal bovine system for at least 2 passages before the experiments. DPSCs and MSCs were cultivated at 37 °C under humidified atmospheric conditions (5% CO2 and 95% air). The pulp variant of MXRA5 tagged with FLAG at the C-terminal (PV-MXRA5-FLAG) was amplified from the DPSC cDNA generated using SSIV (Thermo Fisher Scientific) with reverse primers having the FLAG coding sequence. Then, the amplified PV-MXRA5-FLAG was ligated into pLVSIN-CMV-Pur Vector (Takara Bio Inc., Otsu, Japan) to obtain pLVSIN-CMV-Pur-PV-MXRA5-FLAG, and the sequences were verified. Then, DPSCs stably overexpressing PV-MXRA5-FLAG (DPSC-PV-MXRA5) and control (DPSC-empty) cells were generated as described previously52 (link).
3
Retroviral and Lentiviral Vector Production
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Retroviral vectors expressing SV40LT, EGFP and human K-RasG12D were based on the vector SFG-MCS, a derivative of the plasmid MFG (67 (link)). To generate recombinant retroviruses, we transfected 10 μg viral vector DNA per 6-cm plate into PlatE packaging cells (68 (link)) using the calcium phosphate co-precipitation method. To produce lentiviral particles for PINK1 re-expression, we inserted the human PINK1 coding sequence into the lentiviral vector pLVSIN-CMV-Pur (Takara Biotech) and co-transfected 293T cells in 6-cm plates with 5 μg lentiviral vector plasmid, 3.33 μg HIV gag/pol expression plasmid psPAX2 (Addgene #12260) and 1.67 μg VSV-G expression plasmid pMD2.G (Addgene # 12259) (3:2:1 ratio). Viral particles in the medium were collected 24 and 48 hours after transfection, passed through a 0.45 μm filter and stored at -80°C.
4
Lentiviral Vectors for TMPRSS2 and ACE2
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Human TMPRSS2 and ACE2 genes were cloned into the self-inactivating lentiviral vector plasmids, CSII-CMV-MCS-IRES2-Bsd (RIKEN BRC) and pLVSIN-CMV Pur (Takara Bio), respectively. The resulting constructs were named CSII-CMV-TMPRSS2-IRES2-Bsd and pLVSIN-CMV-ACE2-Pur. For lentiviral vector preparation, 293T cells were co-transfected with the lentiviral vector plasmid and Lentiviral High Titer Packaging Mix (Takara Bio). The culture supernatants containing lentiviral vectors were used to inoculate target cells. Vero cells stably expressing TMPRSS2 (Vero-TMPRSS2) and 293T stably expressing ACE2 (293T-ACE2) were selected in the presence of blasticidin S or puromycin.
5
Generation of mEGFP-expressing Lentivirus
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mEGFP-C1 plasmid was gifted from Michael Davidson (RRID:Addgene_54759). The sequence of mEGFP was subcloned into pLVSIN-CMV Pur (Takara). Lentivirus particles were prepared by the Lentiviral High Titer Packaging Mix with pLVSIN series (Clonetech, Palo Alto, California) using Lenti-X 293 T-cell lines. mEGFP-expressing lentivirus particles and 10 µg/mL of polybrene were added to the media of B16/BL6 cells, and then incubated for 24 hours. mEGFP-transduced cells were selected with 1 µg/mL of puromycin.
6
Lentiviral Expression of TMPRSS Proteases
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The human TMPRSS2, human TMPRSS11D, human TMPRSS11E, and human TMPRSS13 genes were individually cloned into the self-inactivating lentiviral vector plasmid CSII-CMV-MCS-IRES2-Bsd (48 (link)), which was kindly provided by H. Miyoshi (RIKEN BRC, Tsukuba, Japan). Codon-optimized TMPRSS2 and TMPRSS11D genes were designed by the IDT codon optimization tool (Integrated DNA Technologies, Coralville, IA), were synthesized as custom-made DNA fragments (gBlocks; Integrated DNA Technologies), and were cloned into CSII-CMV-MCS-IRES2-Bsd and pLVSIN-CMV Pur (TaKaRa Bio, Kusatsu, Japan), respectively. For the preparation of lentivirus particles, the lentiviral vector plasmid and lentiviral high titer packaging mix (TaKaRa Bio) were cotransfected into Lenti-X 293T cells (TaKaRa Bio) with TransIT-293 transfection reagent (Mirus Bio, Madison, WI). The culture supernatant containing lentiviral vectors was filtered through a Minisart 0.45-μm-pore size filter (Sartorius, Göttingen, Germany) and then inoculated to MA104 cells in the presence of 10 μg/ml Polybrene. To generate T2T11D cells, MA104 cells were inoculated with lentiviral vectors expressing TMPRSS2 and TMPRSS11D and were selected with blasticidin S (for TMPRSS2-expressing cells) and puromycin (for TMPRSS11D-expressing cells).
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