To generate individual lentivirus vectors, HEK293T cells (5 × 105 cells per well in a 6-well plate) were transfected with individual transfer vector (1 μg), pCAG-HIVgp (0.75 μg), pRSV-Rev (0.25 μg), and pCMV-VSV-G (0.25 μg) using TransIT-293 (catalog no. MIR2700; TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. At 24 h after transfection, culture supernatants were filtered through 0.45-μm-pore-size filters (catalog no. 725-2545; Thermo Fisher Scientific). HEK293T cells were transduced with the individual shRNA lentivirus. At 48 h after transduction, knockdown efficiency was determined by Western blotting or RT-quantitative PCR (RT-qPCR).
Transit 293
Manufactured by Takara Bio
Sourced in Japan
TransIT-293 is a transfection reagent designed for high-efficiency, low-toxicity delivery of nucleic acids into mammalian cells, particularly HEK-293 cells. It facilitates the uptake and expression of plasmid DNA, mRNA, and other nucleic acid constructs in these cell lines.
Automatically generated - may contain errors
5 protocols using transit 293
1
Lentivirus Vector Production and Knockdown Efficiency
2
Recombinant SARS-CoV-2 Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To recover recombinant SARS-CoV-2, BACs encoding the full-length SARS-CoV-2 genome were transfected into HEK293T cells by using TransIT-293 (TaKaRa) according to the manufacturer's protocol. At 3 days post-transfection, the supernatant containing viruses was collected and inoculated onto VeroE6/TMPRSS2 at 37 °C to prepare the virus stock. The virus titers of the stock viruses were determined by using plaque assays in VeroE6/TMPRSS2. The stock viruses were subjected to NGS as described17 (link),18 (link) to confirm the absence of unwanted mutations. All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo.
3
HA Subtype Binding Assay
4
Generation of SARS-CoV-2 Infectious cDNA Clones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-genome nucleotide sequence of MASCV2-p25 was assembled into the pBeloBAC11 vector to generate infectious cDNA clones under the control of a cytomegalovirus (CMV) promoter by using Gibson Assembly Master Mix (NEW ENGLAND BioLabs, Ipwich, MA, USA), as described previously [32 (link)]. The sequence encoding the fusion construct Venus-porcine tescherovirus (PTV-1) 2A proteolytic cleavage site was placed upstream of the N gene [33 (link)]. The constructed BAC was introduced into DH10B E. coli (Invitorogen, Waltham, MA, USA) by use of electroporation. The E. coli were amplified at 37 °C and the BACs were extracted using NucleoBond Xtra Maxi (TaKaRa, Kusatsu, Japan).
To recover recombinant SARS-CoV-2, the BAC encoding the full-length SARS-CoV-2 genome was transfected into HEK293T cells using TransIT-293 (TaKaRa, Kusatsu, Japan), according to the manufacturer’s protocol. At 3 days post-transfection, the supernatant containing the virus was collected and inoculated onto VeroE6/TMPRSS2 at 37 °C to prepare the virus stock. The virus titer of the stock was determined using plaque assays in VeroE6/TMPRSS2.
To recover recombinant SARS-CoV-2, the BAC encoding the full-length SARS-CoV-2 genome was transfected into HEK293T cells using TransIT-293 (TaKaRa, Kusatsu, Japan), according to the manufacturer’s protocol. At 3 days post-transfection, the supernatant containing the virus was collected and inoculated onto VeroE6/TMPRSS2 at 37 °C to prepare the virus stock. The virus titer of the stock was determined using plaque assays in VeroE6/TMPRSS2.
5
Immunofluorescence and Immunoperoxidase Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T cells were transfected with each HA-expressing plasmid by use of Trans-IT 293 (Takara bio). At 24 h post-transfection, the cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-100. Antigens were probed with S9-1-10/5-1, CR9114 (Dreyfus et al., 2012 (link)), or 4-6-19/6, followed by Alexa Fluor 488-conjugated donkey anti-human IgG (H + L) (Jackson Immuno-Research) or by peroxidase-conjugated donkey anti-human IgG (H + L) (Jackson Immuno-Research) and SIGMAFAST 3,3′-Diaminobenzidine tablets (SIGMA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!