PEI纳米颗粒基因转染技术方法

1、PEI纳米颗粒基因转染技术方法发布日期:2008-1-8 热门指数:2417ABSTRACTThis protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing upt

2、ake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection using these complexes. The conjugation step involves the use of the s

3、uccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional cross-linker, to form a stable bond between PEI and peptides containingthiol groups.MATERIALSReagentsoo o o o Dc protein assay kitDMEM cell culture medium with 10% fetal bovine serum (FBS)DMEM (Dulbecco's Mod

4、ified Eagle's Medium)100 units/ml penicillin 100 µg/ml streptomycin2 mM glutamine10% fetal bovine serum (FBS)Exponentially growing mammalian cells Lithium chlorideLuciferase assay reagentsDimethylsulfoxide (DMSO)OptiMEM serum-free cell culture mediumPEI polymers (MW 600-1000 kDa, Fluka; MW

5、750 kDa, 25 kDa, 2 kDa, and 800 Da,Sigma-Aldrich; MW 1.2, 10, or 70 kDa, Polysciences) Peptides, prepared using conventional solid-phase, chemical synthesis methodoPhosphate buffered saline (PBS)137 mM NaClo o o2.7 mM KCl 10 mM Na2HPO4 2 mM KH2PO4To prepare 1 liter of PBS(Phosphate-buffered Saline),

6、 dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 ml of distilled H2O. Adjust the pH to 7.4 (or 7.2 if required) with HCl. Add H2O to 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm) on liquid cycle

7、 or by filter sterilization. Store the buffer at room temperature. If necessary, PBS may be supplemented with 1 mM CaCl2 and 0.5 mM MgCl2. Can bemade as a 10x stock. Plasmid DNA encoding the luciferase reporter geneReporter Lysis Buffer, 5X (Promega), dilute to 1X in PBSSuccinimidyl-4-(N-maleimidome

8、thyl)cyclohexane-1-carboxylate (SMCC) Water, ultrapure sterilized Equipment Dialysis membranes (molecular weight size exclusion specification for purification of sidereaction and excess products) Freeze dryerLuminometerMagnetic stirrer and magnetic rodReaction vesselTissue culture vesselsMETHODActiv

9、ation of PEI with a cross-linker1.Prepare a SMCC stock solution of 50 mM in DMSO. SMCC is moisture sensitive. The stocksolution should be prepared using high-quality anhydrous DMSO in a dry nitrogen atmosphere. When stored at 4°C, the SMCC solution remains stable for 3 months. Steps 1-10 should

10、 be performed in achemical fume hood following chemical safe handling procedures.2.Prepare a 10 mg/ml stock solution of PEI in DMSO. Add 2-5 mg of lithium chloride to increasethe solubility of PEI.3. Using a syringe, slowly add the SMCC solution into the PEI solution. Incubate the reaction for2 hour

11、s at room temperature. The amount of SMCC solution added should be based on the desiredmolar ratio between SMCC to PEI.4. Purify the modified PEI by dialysis against ultrapure water for 2 days, changing the water atleast five times a day.5. Collect the solution in a dialysis tube and freeze dry the

12、sample.Conjugation of peptide to activated PEI6.7.8. Prepare a peptide stock solution of 20-50 mM in PBS. Prepare a 10 mg/ml stock solution of the activated PEI (Step 5) in PBS. Slowly add the peptide solution to the activated PEI solution. Incubate the conjugationreaction for 24 hours at room tempe

13、rature.The amount of the peptide added to the reaction should be based on the desired molar ratio in the finalconjugate.9. Purify the peptide-conjugated PEI by dialysis against ultrapure water for 2 days, changing thewater at least five times a day.10. Collect the solution into a dialysis tube and f

14、reeze dry the sample.Preparation of PEI/DNA complexes11.i.ii.iii. Prepare the stock solutions. Prepare a 1 mg/ml plasmid DNA stock solution in ultrapure water. Prepare a stock solution of PEI (Step 5) or peptide-conjugated PEI (Step 10) to contain 10 nmol amino nitrogen per microliter in ultrapure w

15、ater (pH 7.2). For ternary complexes, prepare a 5-mg/ml stock solution of a targeting peptidelinked with a DNA-binding sequence in ultrapure water.Steps 11-22 should be performed under aseptic conditions using sterile reagents. Manipulation of thecomplexes should be performed at room temperature in

16、a horizontal flow hood.12.i. Prepare the working solutions. Dilute 1 µg of plasmid DNA (for transfection of cells seeded in a well of a 24-well plate) in 50 µl of OptiMEM serum-free cell culture medium. Dilute the appropriate quantity of PEI or peptide-conjugated PEI in 50 µl of OptiM

17、EMserum-free cell culture medium.ii. For ternary complexes, dilute the appropriate quantity of the targeting peptide in 50µl of OptiMEM serum-free cell culture medium.13. Add the peptide-conjugated PEI solution dropwise into the DNA solution while vortexing. Or,for ternary complexes, add the ta

18、rgeting peptide dropwise into the DNA solution while vortexing.14. Incubate the mixture for 30 minutes at room temperature. The peptide-conjugated PEI/DNAcomplexes may now be used directly for transfection (Step 16)15. To form ternary complexes, add the PEI solution dropwise into the targeting pepti

19、de/DNAcomplexes while vortexing. Incubate the ternary complexes for 30 minutes at room temperature.The ternary complexes may now be used directly for transfection (Step 16)Transfection assay16. One day before transfection, harvest mammalian cells, grown in DMEM complete cell culturemedium with 10% F

20、BS by trypsinization, and replate them into 24-well plates at a density of 50,000cells/well. Incubate the cultures for 24 hours at 37°C in a humidified incubator with 5% CO2.17.18. Remove the medium from the wells and wash the cells twice with PBS (prewarmed to 37°C). Add the following to

21、the cells, and incubate them for 4 hours at 37°C in a humidified incubatorwith 5% CO2. 100-150 µl/well of OptiMEM serum-free cell culture medium 100-150 µl/well of the genetransfection complex containing 1 µg of plasmid DNA (Step 14 or Step 15)19. Remove the transfection solution

22、, wash the cells twice with PBS (prewarmed to 37°C) andadd 1 ml/well of DMEM complete cell culture medium with 10% FBS. Incubate the cells for 24-48hours.20. To assay for luciferase expression, lyse the cells by adding 100 µl/well of 1X Reporter LysisBuffer (dilute 5X stock solution with P

23、BS).21. To detect luciferase activity, add 20 µl of cell lysate to 100 µl of luciferase assay reagent.Measure luciferase activity with a luminometer.22. To normalize the luciferase activity, determine the protein concentrations of the cell lysatesusing the Dc protein assay kit.ACKNOWLEDGMENTSThe work was fund