Gene transfer into mammalian cells using targeted filamentous phages
Gene transfer into mammalian cells using targeted filamentous phages
Because of their genetic simplicity, phages are commonly used in vector genetic optimization approaches for therapeutic gene transfer. In addition, since phage propagation is strain-specific, different hosts are equally suitable for genetic modification, modification, or even for optimization of yield, genetic stability, preparation, and expense using genetic screening. The choice of targeting ligand determines the specificity of the targeted phage transduction. Since proteins can be efficiently expressed and are biologically active after being secreted into the peripheral space of the bacterial cytoplasm and into phage particles, genetic targeting is largely restricted to proteins . Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Gene transfer into mammalian cells using target-directed bacteriophages Move Gene transfer into mammalian cells using target-directed bacteriophages Materials reagents β-Galactosidase Dimethyl sulfoxide (D M S O ) solution of camptothecin (l ○ m m o l /L ) (Sigma-Aldrich C 9911) -20°C Storage. Cell culture medium: R P M I 1640 + 10 % fetal bovine serum (FBS), O.l m m o l /L non-essential amino acids, l m m o l /L sodium pyruvate, 2 m m o l /L L-glutamine and S O p g / m l gentamicin. D N a s e I ( G I B B O 18068-015) edta (0-5m o l /l ) Fixation buffer (p B S containing 0.925% formaldehyde, 0.02% sodium azide and glucose, p H 7. 4) Glycerol (20 %) Spade host strain (Stratagene 200249 X L l -B l u e receptor cells) The P B S solution was prepared via basal urea (l. 0 m o l /L ) < ! > The cells were stored in PBS solution (Sigma-Aldrich H 8 6 27 ) at 20 °C. L B medium plates (1 % tryptone, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract) 5 % yeast extract, 0.5 % NACL, PH 7.0 and 2 % agar). L B + medium plates (1 % tryptone, 0.5 % yeast extract, 0.5 % NaCl, PH 7.0 and 2 % agar). 5 % yeast extract, 0.5 % NaCl, pH 7.0 and 2 % agar) 5 % NACL, pH 7.0, 2 % agar and 60ug/m l ampicillin. M g C l 2 (l m o l / L ) P C-3 cell line (N C I cell bank) and H T 1229 (A T C C ) Phosphate buffer (P B S ) P B S + A E B S F (0- 2m m o l /L ) (4- [2-aminoethyl] benzenesulfonyl fluoride) (R o c h e 1585916) Complex M 1 3 Phage D N A SOC Culture Base (GIBCO 15544-034) Sodium chloride (NaCl, I.5m o l /L) / 30 % polyethylene glycol (PEG) 8000 Targeted phage particles containing the reporter gene GFP Triton X-114 (10 %) Trypsin (0.25 %) 2 5 %) ( G I B C O 25200-056) UV irradiation (Stratalinker UV crosslinker, Stratagene) Instrument Centrifuge bottles Freezer Tubes 37°C and 42°C incubators Microcentrifuge tubes (Eppendorf) Micropipettes Vibration Shaker Syringe filter (0. 45um ) Syringe Tissue Culture Tray (12-well) Tubes (sterile, Falcon 2059) 55°C water bath Method D N a s e I ( G I B B O 18068-015) edta (0-5m o l /l ) Fixation buffer (p B S containing 0.925% formaldehyde, 0.02% sodium azide and glucose, p H 7. 4) Glycerol (20 %) Spade host strain (Stratagene 200249 X L l -B l u e receptor cells) P B S solution via basal urea (l. 0 m o l /L ) (Sigma-Aldrich H 8 6 2 7 ) L B medium plates (1 % tryptone, 0.5 % yeast extract, 0.5 % NACL, PH 7.0 and 2 % agar). 5 % yeast extract, 0.5 % NACL, PH 7.0 and 2 % agar) L B + medium plates (1 % tryptone, 0.5 % yeast extract, 0.5 % NaCl, PH 7.0 and 2 % agar). 5 % yeast extract, 0.5 % NaCl, pH 7.0 and 2 % agar) 5 % N a C l , pH 7.0, 2 % agar and 60 M g/m l ampicillin) M g C l 2 (l m o l / L ) P C-3 cell line (N C I cell bank) and H T 1229 (A T C C ) Phosphate buffer (P B S ) P B S + A E B S F (0- 2m m o l /L ) (4- [2-aminoethyl] benzenesulfonyl fluoride) (R o c h e 1585916) Complex M 1 3 Phage D N A SOC Culture Base (GIBCO 15544-034) Sodium chloride (NaCl, I.5m o l /L) / 30 % polyethylene glycol (PEG) 8000 Targeted phage particles containing the reporter gene GFP Triton X-114 (10 %) Trypsin (0 . 2 5 % ) ( G I B C O 25200-056 ) 2 5 % ) ( G I B C O 25200-056) UV radiation (Stratalinker UV crosslinker, Stratagene) 2 X Y T broth medium (1.6 % tryptone, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract). 0.5 % yeast extract, 0.5 % NACL, pHCL 5 % NACL, p H 7.0 and 60ug/ml ampicillin). Instruments Centrifuge bottles Freezer Tubes F A C S / Flow Cytometer with Fluorescein Isothiocyanate (F I T C ) Filtration Device 37°C and 42°C incubators Microcentrifuge tubes (Eppendorf) Micropipettes Vibration Shaker Syringe filters (0. 45(um )) Syringe Tissue Culture Tray (12-well) Tubes (sterile, Falcon 2059) 55°C water bath Method Transformation of bacteria Replicative recombinant phage vectors modified to contain mammalian expression cassettes were used to transform host bacteria using standard molecular biology techniques. In our study, a commercially sourced C M V promoter and the growth hormone (G H ) polyadenylate D N A were used. 1. melt IOO m I receptor cells on ice. 2 . Add I.7m1 p-galactosidase to the cell suspension and incubate on ice for l O m i n . 3 . 50n g of replicative phage D N A was mixed with the cells and incubated on ice for 30m i n . 4. Heat-excite the bacteria at 42°C for 45s and place the bacteria on ice for 2m i n . 5. Add 900M SOC medium to the cells and incubate at 37°C with 250r/m i n vibration for l h . 6- Coat the cells with L B + culture plate and incubate at 37°C overnight. Preparation of targeted phage particles Using standard phage display techniques, the desired ligands are modified on the gill so that the final particles will display ligand-p i n fusions that can rake phage onto mammalian cells. 7. Cultivate the contaminated bacteria with 2X Y T medium containing 6ug/ml ampicillin at 3r C with 300r/m in shaking and incubate the bacteria overnight. 8. Centrifuge the bacterial cultures at 6000 g at 4°C for l0 m i n . 9- Retain the supernatant and add 1/5 volume of pre-cooled 1.5m o l/L NACL/30% PEG. Mix well and incubate on ice for 2 h to precipitate phage. 10.4°C, 15 OOOg ■ Centrifuge for 30 m i n to collect the phage, remove the supernatant and aspirate any residual liquid. 11. Resuspend the phage in a PBS solution containing 0-2 mMol/L AEBSF and allow to stand at 4°C for 30 m i n . 12.20 OOOg ■ Centrifuge for 20-30 m i n to remove residual lice. 13-If further concentration of phage is required, repeat steps 9 to 11. 14- Add M g C l z to a final concentration of lO^m o l /L with 6 units of D N a s e I per ml of phage solution. leave at room temperature for 20m i n and terminate the reaction with IOuI ○.5m o l / L E D T A per ml of phage solution. 15- Immediately add 1/5 v/v pre-cooled 1.5 m o l / L N a C l / 30 % P E G . Mix well and place on ice for ^ 2 h to allow the pycnidia to settle. Centrifuge for 30 m i n at 16.4°C, 15 o o o g. Collect phage, remove supernatant and aspirate all residual liquid. 17. Resuspend the phage in PBS solution containing 0.2m m 〇 I/L AEBSF, and leave at 37°C for 5 to 15 m i n ^ generation for 30m i n o . 18. Centrifuge at 20 to 30 m i n as hard as possible at 20,000 to remove the residue. 19. Filter the phage through a 0.45um filter, freeze with 20% glycerol, and store at 70℃. Endotoxin is removed by Triton X-IM phase separation. Removal of endotoxin is critical prior to any cell and animal studies. 200 - Add 1(%1 10% Triton X-m per mL of sample and place on ice for 30m i n , shaking occasionally. Let stand at 37°C for IOmin0 21 - Centrifuge at 14,000r/m i n for lOmin at room temperature, retaining the aqueous phase (upper layer). 22. Repeat the phase separation (steps 20 and 21) twice. Titrate the phage and use the phage spots to form units to determine the yield. 23. Preheat L B plates at 37°C, melt the top layer of agar (L B) and place in a 55°C water bath. 24- Incubate F ' bacteria to 〇 D s. = 0. 5, and 3(%1 cells were taken into each Falcon 2059 tube to be tested. 25- Perform a gradient dilution with P B S at a starting dilution of 1,000-fold (5^1 dilution into 500^1 P B S) and repeat each dilution several times. Remove IOOuI from each dilution tube and add to bacterial cells. 26. Add 3 m l of agar to each tube, mix well and pour onto the top layer of a preheated L B plate, allow the top layer of agar to solidify and then invert at 37°C overnight. 27.4 Count the phage spots by tens and multiply the number of spots, the dilution and the volume of the inverted plate to determine the titer (pfu/m l). Transduction of cultured cells 28- Inoculate I m l cell cultures in 12-well tissue culture trays and incubate overnight at 37°C in a 5 % C O 2 oven. Plate density is determined by the growth rate of the cells. After overnight incubation, the cells should reach 25% confluence in the 12-well plate, and we inoculated PC-3 cells at a density of 2XIO4 cells/well. 29. Discard the cell culture medium and replace it with phage-containing medium. The typical dose of targeted phage for maximum transduction efficiency is IO11P f u A n L 5 % CO2 incubated at 37°C for 24-96 h. If genotoxic treatment is used, the dose of phage is not sufficient for the cell culture. If a genotoxic treatment is used, incubate for 40 h . 30. Remove the phage-containing cell culture medium and wash with PBS to collect cells for reporter gene analysis. Add 150ul of trypsin and let the cells stand at 37°C for 2 to 3 m i n to detach the cells from the culture plate. Immediately add 350M 1 fixation buffer. Analyze cells by flow cytometry with FITC filter. Genotoxicity Treatment Contraceptive organisms are added to the culture and the genotoxicity treatment is initiated 40 h later. The treatment conditions should be optimized for each target cell line. 31. For heat stress and UV radiation, cell culture medium containing 10 % fetal bovine serum is required. For heat stress and UV radiation, cell culture medium containing 1 0 % fetal bovine serum is required. Instead, 1 to l O O um o l /L hippocampus minus or 10-100 m m o l /L light basal vein should be added. 32. Remove phage-containing medium. Replace with genotoxic medium as above, or with medium containing 1 0% F B S for heat stress or U V radiation. 33. Incubate directly at 37°C or 42°C (heat stress) or irradiate (100~lOOJ/m2 ) for 7 h. 34. Wash with PBS 3 times. 35. Add fresh medium containing 10% FBS. Incubate at 37°C for 24~48 h. 36. Wash with PBS three times and test the genotoxicity treatment effect by direct observation of GFP or FACS analysis. For more product details, please visit Aladdin Scientific website.
2 X Y T broth medium (1.6 % tryptone, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract, 0.5 % yeast extract). 0.5 % yeast extract, 0.5 % NACL, p H 5 % NACL, p H 7.0 and 60ug/m l ampicillin.
F A C S / Flow Cytometer with Fluorescein Isothiocyanate (F I T C ) Filtration Device