SIV Vectors as DNA Delivery Mediators
SIV Vectors as DNA Delivery Mediators
Provides some methods on how to use the miniature SIV packaging system and SIV gene transfer vectors to obtain high titer and safe lentiviral vectors developed from S IV. Author: T. Friedman et al, Translator: Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Production of SIV vector particles for gene transfer applications Move Production of SIV vector particles for gene transfer applications Materials carve on trial basis Bovine serum albumin (B SA component 5) 1 % BSA dissolved in PBS is used as a freezing medium and stored at 4 °C. Calphos Mammalian Transfection Kit for Calcium Phosphate Transfection Transfection and transduction of cell lines 293T cells T E 671 cells (human rhabdomyosarcoma, ATCCCRL-8805) sM AG I short-tailed ape cells These cells should allow SIVmac251 replication and possess a stably integrated HIV-I LTR-driven ZacZ transcription unit induced by SIV/HIV M. The cells should have a ZacZ transcriptional unit with a ZacZ transcriptional unit. The sMAGI cell growth medium is DMEM/10% FCS/Thymidine (50ug/ml)/G418 (200ug/ml). DM EM medium containing 0.l l g/L potassium vitamin B 6 and vitamin B 6 was used as the growth medium. Add 10% FCS, 100 ug/ml streptomycin and lOOp/ml penicillin in DMEM and store at 4°C. Inactivated fetal calf serum (FCS) G418 (100 mg/ml dissolved in water, Geneticin; Invitrogen) Filtered through a 0.22 um pore size membrane and stored at 20°C. Glutaraldehyde solution (0.5% in PBS, stored at 4°C). Thiomycin B dissolved in PBS (50m g/m l, stored at 4°C, Invitrogen) Nucleic acid Lentiviral vector DNA with a selective internal promoter encoding a slow self-deactivating vector and encoding the The selection of promoters in the internal transcription frame should be based on the characterization of the promoter in the relevant raking cells. Envelope glycoprotein expression vectors (e.g. VSV-G) Viral structural proteins (Gag-PoD expression vectors) paraformaldehyde solution (4 % in PBS, stored at room temperature) Sterile PBS solution free of calcium, magnesium and sodium bicarbonate Polyglutamine storage solution (bemecium bromide; 8m g/m l dissolved in H2O; Sigma-Aldrich) Trypsin Trypsin-E D T A dissolved in a sterile I X Hank equilibrium free of calcium and magnesium Vilene 1 : 5000 (Invitrogen) X-gal Buffer 43.5 m m ol/L sodium deoxycholate (compound monohydrate; Sigma-Aldrich) 2 mmol/L MgCl2 - 6 H2O (Sigma-Aldrich) 5 mmol/L potassium ferrocyanide (Sigma-Aldrich) 5 mmol/L potassium hexacyanide (Sigma-Aldrich) 0.002% NP-40 (RocheDiagnostics) Dissolve in PBS. For sM AGI cells, p H was adjusted to 9.0 with N aO H X-gal (5-bromo-4-chloro-3-indolyl-(3-1>galactoside) (Euromedix) Prepare a 60 X storage solution and dilute with (DMF; Sigma-Aldrich) to a final concentration of 40 mg/ml. store at -20°C. Add X-gal to fresh X-gal buffer before staining cells. Equipment FACS Calibur Flow Cytometer (Becton Dickinson) Flow Tube (Becton Dickinson) Fluorescence Microscope Filter membrane (0.45um pore size) Reverse transcriptase assay kit (Roche Diagnostics) Standard tissue culture equipment, including 100mm print and six-well petri dishes Ultracentrifuge tubes (26 ml, 25 mmX89 mm, polyisotope, Beckman) Methods Production, Concentration and Preservation of S IV Vector Particles 1. The day before transfection: Spread approximately 2. 4X106 293T cells into IOOmm diameter tissue culture dishes, maintaining a final volume of 12 ml/dish. 2. On the day of transfection: Transfect 8. Iug S IV packaging vector, 8. Ijug transfer vector and 2.5ug envelope expression vector by calcium phosphate method. 3. The second day of transfection: After incubation for about 15 hours, gently remove the medium from the petri dish and replace it with IOml of fresh medium. 4. Transfection day 3: After incubation for about 36 h, collect the virus: centrifuge the medium at low speed to remove the cell residue, and filter with a 0.45um membrane. The filtered supernatant can be stored at 1.80°C. The virus can be collected by centrifugation at low speed. 5. Transfer the supernatant to a 26 ml ultracentrifuge tube. Collect the viral pellet by centrifugation at 110 OOOg (32,000r/min) for 2 h at 4°C using a 70Ti Beckman centrifuge. 6. Remove the supernatant and wash the walls of the tube with 5 ml of PB S. Invert the tube onto filter paper. The tubes are inverted on filter paper for 5 min, allowed to dry, and the walls are wiped dry. 7. Resuspend the virus pellet in ice-cold PB S containing 1% BSA by adding 1/100th of the volume of the initial viral supernatant and ice bath for 2 hours. 8. Aliquot the virus pellet into microcentrifuge tubes and store in 80° C or liquid nitrogen. Transduction Analysis 9. The day before: Inoculate TE671 or sMAGI target cells into 6-well plates at a density of 4 X 105 cells/well with a final volume of 2 ml/well. 10. The day of: Prepare a series of carrier dilutions with lml DMEM/10% FCS and add to cells at 6ug/ml polyglutamine. 11. Incubate the medium at 37°C for at least 4 h. Aspirate off the carrier-containing medium and replace it with 2 ml of fresh medium. Incubate the cells at 37°C for 72 h. Incubate the medium at 37°C for at least 4 h. Blot out the medium containing the carrier and replace it with 2 ml of fresh medium. 12. Day 3: Digest cells with trypsin, fix with 4% paraformaldehyde in PBS for 30 min and transfer to flow tubes. Cells were analyzed by flow cytometry. When analyzed, the efficiency of transduction is usually determined by the percentage of GFP-positive cells after transduction of 4 X IO 5 target cells with Immunovirus supernatant. The titer of infection is expressed in transfection units A nl and can be calculated using the following formula: where d represents the dilution factor of the viral supernatant represents the percentage of G FP positive cells measured by flow analysis with the viral supernatant required to transduce 5 % to 10 % of G F P positive cells. The multiplicity of infection (moi) is the ratio of infected viral particles to target cells. Safety testing of vectors: detection of PCR 13. The day before: inoculate sMAGI target cells into a 6-well plate at a density of 4X 105 cells/well with a final volume of 2 ml/well. 14. Day: sMAGI cells are initially infected with S IV vector particles. lml DMEM/10% FCS is prepared as a series of vector dilutions and added to the cells in the presence of 6ug/ml polyglutamine. After 4 h of incubation, the carrier-containing medium was aspirated and replaced with 2 ml of fresh medium. The cells were incubated at 37°C for 72 h. The cells were then incubated for 4 h in a series of carrier dilutions containing 6ug/m l of polyglutamine. 15. Day 3: Digest the cells with trypsin. a- Detection of the percentage of G F P positive cells: 1/3 of the cells were fixed with P B S containing 4 % paraformaldehyde for 30 min and then the cells were analyzed by flow cytometry. b. Detection of β-galactosidase expression: re-inoculate the other 1/3 of the cells into a 24-well plate with a volume of lm l. When the cells are full grown, fix them for lOmin with Iml PBS containing 0.5% glutaraldehyde, then wash them again with Iml PBS, and incubate them for 2 h at 32°C with X-gal solution (pH 9.0). c. Allow final PCR to diffuse: Replant the last 1/3 of the cells into a 6-well plate with a volume of 2 ml per well. incubate the cells for 10-15 days. 16. Day 14: One day before the 2nd infection, inoculate sMAGI target cells into 6?L plates at a density of 4X105 cells/well with a final volume of 2 ml/well. 17. Day 15: Infect sMAGI cells as target cells with supernatant (from 13 to 18d of infection medium) from the initial infection of target cells. Infect target cells with Im l of supernatant for 4 h at 37°C. Replace with 2 ml of normal medium. After infection, the cells were incubated at 37°C for 48 to 72 h. The activities of reverse transcriptase and GFP and β-galactosidase were assayed. To demonstrate the presence of G FP-containing SIV vectors for movement, show that the SIV reservoirs do not contain PCR and M recombinant reverse transcription viruses, and that neither G FP nor β-galactosidase should be expressed at 48-72 h post-infection in all experiments. In addition, the reverse transcriptase should remain inactivated in the supernatant during the primary and secondary infection of the cells. As a positive control in the assay, sMAGI cells can be infected with wild-type SIVmac251, and then the activity of the reverse transcriptase enzyme and the ability to express genes such as tat can be examined after the recombinant retrovirus undergoes replication and amplification during this time period. For more product details, please visit Aladdin Scientific website.
the reporter gene GFP
Samples can be stored for at least 6 months without affecting the viral titer.
Titer= (4X 105/l0 ) Xd