Efficient DNA delivery by phage into mammalian cells
Efficient DNA delivery by phage into mammalian cells
Transferring genes into cells and expressing new proteins, or in this way altering the expression levels of endogenous proteins, can modulate the physiological functions of cells. This approach is very useful for the study of cellular functions and also has good application prospects in the treatment of a large number of diseases. Therefore, it is important to develop novel and efficient non-viral D N A delivery systems. Recently, some cationic short skins known as peptide/protein transduction domains (P T D ) have been found to efficiently deliver a wide range of substances, including D N A, into a variety of cells. This chapter describes the surface-presenting H I V -1 T A T P T D X phage, which is not cytotoxic per se but can efficiently deliver plasmid D N A into a large number of different cells in a concentration-dependent manner. Author: T. Friedman et al, Translator: Jingwei et al. This experiment is from "Gene Transfer".
Operation method
Delivery of DNA to mammalian cells with X phages exhibiting T A T transduction domains Move Delivery of DNA to mammalian cells with X phages exhibiting T A T transduction domains MATERIALS reagents Cesium chloride Chloroform D A P I (4,,6-diamidine-2-phenylindole H C l DNaseI (Sigma-Aldrich) H-SM Buffer (contains l O m m o l / L H E P E S ^ N a O H solution, l O m m o l / L M g S O 4 solution, l O O m m o l / L NaCl solution, p H 7.5) L E 3 9 2 Cells Fluorokinase Test (P r o m e ga) Lysogenic Esc/ieric/iia coZi, human D 1180-GFP, or D 1180 Brilliant Fluorophore Enzyme Culture base DMEM medium, containing 10 % fetal bovine serum, is used for the culture of COS^l, 293, A 431, NIH-3T3 cells. MEM culture medium containing 10 % fetal bovine serum for cell culture of WI 38/VA 13/2RA, HELLA LB Culture Base, containing 10 g/L pancreatic peptone (Becton Dickinson), 5 g/L fermented yeast extract (Becton Dickinson), 10 g/L NACL, 10 mMoL/L MgSO4, 10ug/ml ampicillin. Phage Chromogen D N A (10n g, purified, 2. 5 X l O 8 copies) Plasmids: pTrc-D, pTrc-T A T -D, pTrc-R G I > D, pTrc-V N -D, pTrc-N L S --D, constructed from pTDrcHis A (Invitrogen). Phage Different types of recombinant phages (2.5X 108pfu ) TAT phage (2.5 X 106pfu or 2.5 X 109pfu ) Wild-type phage (2.5 X 1010pfu ) Instrumentation Dialysis Bags fluorescence microscope G F P A cube and W U cube (Olympus Optical C o .L t d , T o k y o , Japan) class chamber slide Incubators at 32°C, 37°C, 38°C and 45°C 2 4-well plates Method Preparation of recombinant X phage Plasmid-transformed lysogenic E. coli were incubated with LB (containing 10 m m o l /L M g S O 4, 100/ng/m L 麵节西林) at 1.32°C until an absorbance value of 0.3 was reached at OD m d . 2. Incubate the bacteria at 45°C for 15 m i n , then at 38°C for 180 m i n . 3 . Treat the bacteria with bromide (final concentration 10 %) and D NASE I (final concentration IOlUgAnl) and harvest the phage particles. 4 - Two rounds of cesium chloride equilibrium centrifugation were used to isolate the phage pellets, which were then purified by dialysis in H-SM buffer. 5. Using LE392 cells as host cells, the titer was determined at 37°C by Plaqueassay. Induction of marker gene expression with recombinant phage 6- The cells to be transfected were inoculated in 24-well plates at a density of 2.5X 104 cells per well and incubated for 2 h. The cells were then incubated for 2 h in a 24-well plate. 7- Wash the cells once with medium and incubate the cells with 500ul of medium containing 2.5 X IO8P fu recombinant phage or IOng purified phage chromosomal DNA for 6 hours at 37°C. 8- Wash the cells twice with medium and continue incubation for 48 h. 9- Cells were collected and quantitatively analyzed for fluorokinase activity using a fluorokinase assay kit, and the results were expressed as mean relative fluorescence units (RLU) with standard deviation. In situ detection of T A T -phage-mediated G FP expression in cultured cells 10. COS^l cells were inoculated in glass chamber slides at a density of 2. 5XIO4 cells per well and cultured for 12 h. The cells were then incubated in a glass chamber slide for 12 h. The cells were then incubated in a glass chamber slide for 12 h. 11. Wash the cells once with medium, and then incubate the cells with 500ul of medium containing 2.5 X IO10pfu or 2.5 X 109pfu of TAT phage or 2.5 X 1010pfu of wild-type phage for 6 hours at 37°C. 12. Wash the cells twice with the medium and continue incubation for 48 h. 1 3 . The cells were collected, the nuclei were stained with D A P I and the expression of G F P was observed by fluorescence microscopy. For more product details, please visit Aladdin Scientific website.
Cell lines: CO&L, 293 (purchased from RIKEN Cell Bank, Wako, Saitama, Japan), A 431, NIH-3T 3, W 138/V A 13/2R A (purchased from the Health Science Research Resource).
B an k , T o k y o , Japan), and H e L a