The lentiviruses were prepared and used in a similar way as previously described (44 (link)). Briefly, lentiviruses expressing CRE-RLuc or indicated shRNA were prepared from HEK 293T cells by co-transfecting lentiviral expression plasmids along with packaging vectors using the calcium-phosphate method (TakaRa). DTC-1 cells were transduced with lentiviruses expressing CRE-RLuc. After puromycin selection, the cells were used directly for reporter assays.
Calcium phosphate method
Manufactured by Takara Bio
Sourced in Japan
The calcium phosphate method is a laboratory technique used to transfect cells with foreign DNA or RNA. The method involves the formation of a calcium phosphate-nucleic acid coprecipitate, which is then added to the cell culture. This allows the genetic material to be taken up by the cells.
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6 protocols using calcium phosphate method
1
Lentiviral Transduction of DTC-1 Cells
2
Engineered Exosome Producer Lentivirus
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The exosome producer third-generation Lentivirus plasmid was custom built by VectorBuilder. Inc., which contained a hPGK1 promoter driving expression of CD63-L7Ae and Connexin43 S368A and EF1α promoter driving ZFP362-PAM- C/Dbox expression. The inclusion of protease cleavage site P2A between CD63-L7Ae and Connexin43 S368A ensured protein separation. Mutations in the bovine growth hormone polyadenylation (bgh-PolyA) transcription termination signal after CD63-L7Ae and Connexin43 S368A enable read-through and expression of complete lentiviral genomic RNA expressed from the RSV promoter. The faulty poly (A) signal allowed for CD63-L7Ae and Connexin43 S368A expression. To obtain pLV-EXOtic-ZFP, DNA fragment between Afe1 and Acc65I i.e., PAMt was removed, overhangs filled in using Klenow Fragment (NEB) and re-ligated. To build the pLV-EXOtic-nLuc vector, the pLV-EXOtic-ZPAMt vector was digested with NsiI and BstBI (NEB) to remove the ZPAMt and replaced with the nLuc gene using NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer’s instructions. The calcium phosphate method (Takara) was used to transfected HEK 293 T cells with a mixture of genome transfer plasmid and packaging plasmid: pRRE, pCMV.Rev, pMD2.G61 (link), at a ratio of 4:2:1:162 (link). The lentivirus vector was used at an MOI of 5 to transduce Mesenchymal Stem cells via spinoculation.
3
Primary Culture of Hippocampal Neurons
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Primary culture of hippocampal neurons was performed as previously described with some modifications (25 (link),30 (link)). The hippocampus of wild-type C57BL/6 mice (Japan SLC, Hamamatsu, Japan) at embryonic day 17 was dissected, and neurons were cultured in glass-bottom dishes (MatTek, Ashland, MA) in culture medium (NbActiv4; BrainBits, Springfield, MA), as described previously (25 (link)). After culture for 4–7 days, the neurons were transfected with the plasmid vector for green-fluorescence-protein-fused synaptotagmin (31 (link)) using the calcium phosphate method (Takara Bio, Shiga, Japan). All animal experiments complied with a protocol approved by the Institutional Animal Care and Use Committee, Tohoku University (2016EgA-003, 2019EgA-001).
4
Efficient VSVG Pseudotyped Virus Production
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For VSVG‐pseudotyped virus production, 293T cells were transfected using the calcium phosphate method (Clontech) using pMLP along with packaging and structural vectors VSVG and GAG/POL. Supernatants containing virus were then used to transduce target cells in the presence of 8 μg/ml polybrene for three rounds of infection. Successfully transduced cells were selected either with puromycin (3 μg/ml) for pMLP infected cells, or were sorted for GFP expression by flow cytometry for GFP‐Luciferase used to infect OVCAR8 cells for bio‐luminescent imaging and was a gift from B. Huang (MIT).
5
VSVG-Pseudotyped Virus Production and Transduction
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For VSVG-pseudotyped virus production, 293T cells were transfected using the calcium phosphate method (Clontech) using either pMLS-tomato (for shRNAs in KP7B cells) along with packaging and structural vectors VSVG and GAG/POL. Supernatants containing virus were then used to transduce target cells in the presence of 8 µg/mL polybrene for three rounds of infection. Successfully transduced cells were sorted for tdTomato expression by flow cytometry for pMLS-tomato infected cells. GFP-Luciferase was used to infect KP7B cells for bioluminescent imaging and was a kind gift from Bonnie Huang (MIT).
6
Lentiviral Vector Production and Titration
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To produce pseudotype virus, the lentiviral SIN vectors were cotransfected with three helper plasmids: gag/pol helper plasmid, the HIV-1 Rev plasmid, and the VSV-G envelope plasmid. The four-plasmid system was transfected into HEK293T cells by calcium phosphate method (Clontech, Mountain View, CA), as described previously.80 (link) For small-scale production, virus-containing media (VCM) was collected 24 and 48 hours posttransfection and pooled. VCM was concentrated by ultracentrifugation (25,000 rpm) over a 20% sucrose solution. Large-scale virus was produced in a manufacturing facility under either GLP or GMP conditions, based on a method published previously.81 (link) VCM was purified by Mustang Q followed by concentration using tangential flow filtration filters.
Lentiviral vectors were diluted (1/8–1/512 for unconcentrated samples or 1/10–1/30,000 for concentrated samples), and 1 ml used to transduce 1 × 105 293T cells in a 12-well plate. Seventy-two hours posttransduction, cells were analyzed for enhanced green fluorescent protein expression or stained with 2F5 for cell-surface expression of C46. Samples were transduced in duplicate.
Using the percentage of positive cells, titer was calculated according to the following formula:
MOI was calculated by dividing the number of virus particles by the number of cells.
Lentiviral vectors were diluted (1/8–1/512 for unconcentrated samples or 1/10–1/30,000 for concentrated samples), and 1 ml used to transduce 1 × 105 293T cells in a 12-well plate. Seventy-two hours posttransduction, cells were analyzed for enhanced green fluorescent protein expression or stained with 2F5 for cell-surface expression of C46. Samples were transduced in duplicate.
Using the percentage of positive cells, titer was calculated according to the following formula:
MOI was calculated by dividing the number of virus particles by the number of cells.
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