Screening, isolation and characterization of target peptides for ligand-directed gene delivery
Screening, isolation and characterization of target peptides for ligand-directed gene delivery
Functional vascular mapping of tissue-specific and angiogenesis-related receptors has been more widely promoted and optimized by isolation of receptors, cell lines, screening techniques for experimental animal models, and isolation techniques for ex vivo and in vivo clinical samples and samples for direct patient use. Recently, a new class of ligand-directed receptors for hybridization of adeno-associated viruses (A A V ) and phages has been designed for the transfer of target genes to cell-surface receptors that are reachable in a systemic delivery manner. Ultimately, these advances could have clinical applications. Author: T. Friedman et al, Translator: Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Screening of in vivo randomized phage display libraries Move Screening of in vivo randomized phage display libraries Materials reagents anesthetic B A L B /c Mice (2 months old) Despite the low cost-effectiveness, nude mice are still the best choice for minimizing cross contamination with fur-associated bacteria. The receptors for most of the isolated ligands differ in different mouse strains. D M E M Tissue Culture Solution K 91 kan bacteria Any other F-hair-positive M - Zi bacteria can be used for contempt amplification. Kanamycin L uria b ro th medium N Z Y b r o th medium Phage display randomized peptide library The generation and production of phage display ZTC randomized polypeptide libraries has been ubiquitous (Smith and Scott 1993; Koivunen etal. I" 9 ). Several libraries in different vectors have been commercialized. Suitable libraries are composed of more than IO8 different phage peptide sequences. In general, peptides less than 9 free residues in length that produce better affinity ligands are preferred for selection. Larger libraries of interpolated sequences are too diverse to be useful for practical applications. Each step after in vivo targeting is particularly critical for quality control of phage libraries. Amplified libraries (as opposed to primary libraries) are not always usable if the absence of inserts or mutations in the phage clones is taken into account. Polyethylene glycol (P E G )/sodium chloride N a C l Dissolve IOOg PEG8000 and IlOg NaCl in 450 ml of water, shake vigorously, sterilize in a pressure chamber, and shake while cooling. Phosphate buffer solution (P B S ) c o c k ta il protease inhibitors (R oche) T B supplement Dissolve 11. 55 g KH2PO4 and 105 g K2 HPO4 in 500 ml H2O and autoclave. Dilute with TB at the ratio of I=10 before use. Terrific Broth (T B) Medium Tetracycline Tris buffer salt solution (T B S ) Instrumentation Sleeve (butterfly I V 23-gauge blue) Cell Culture Flasks Glass Grinder Luria B r o t h Agar Flatware Shaker (37°C) Surgical tools Syringe Methods Injection of phage display libraries 1- Dissolve the selected phage library in DMEM or PBS to a concentration of 3.3 X 1010. transduction units (TU)/ml. 2 . Inject l0 10TU of phage library into the test animal through tail vein. The volume should not exceed 300 ul. 3 . Animals should be kept alive for more than 3~5 min to facilitate the dissemination of the library in vivo. Transcardiac perfusion and washing Transcardiac perfusion of experimental animals must be performed prior to surgical collection of the target organ, which reduces the recovery of nonspecific background phage. However, perfusion of some organs (e.g., kidneys) can increase the aggregation of nonspecific background phage, resulting in poorer experimental results. In addition, perfusion is not recommended in the first round of screening because excessive processing can counteract the binding ability of the peptide, especially when the number of bound peptides is small. To minimize the amount of background phage recovered from blood, mice can be bled completely if perfusion is not done. 4- Make sure that the experimental animals are dissected under deep anesthesia. 5 . Incise the epidermis to the diaphragm to expose the chest and abdominal walls. 6.Open the peritoneum below the sternum without destroying the liver, heart, and other larger blood vessels. 7- Along the coastal arch, cut from below. 8 . Cut the thoracic cavity along the measured line, from the diaphragm up to the axilla, taking care not to destroy the lungs. 9.Turn the front chest wall over to expose the heart. Secure with clips. 10. A syringe with cannula containing approximately 5 ml of room temperature DMEM is injected into the left ventricle. 1 1 . Make a small incision in the right atrium to serve as an outlet for the blood. 1 2 . Perfuse at low pressure to prevent damage to the blood vessels. A total of 50 ml is perfused through the heart. The relative stringency is directly related to the volume used, and a small perfusion volume may be used for the first screening (if perfusion is used). 13. Surgically obtain the target organ and at least one control organ (e.g., lung or brain). Place immediately on ice to avoid internalization of bound phage. 14. Weigh the organs and grind them well with a glass tissue grinder. 15. Add Im I frozen trypsin inhibitor-conjugated DME to the tissue milling solution, vortex, and centrifuge at 4°C, 3000 rpm for 4 m i n. Remove the supernatant. Remove the supernatant. 16. Repeat step 15 (wash 3 times, or once or twice for the first screening). 17. After the last centrifugation, remove the supernatant and keep the sample on ice until the bacterial solution is added. 18- A streak was picked from a K 9U a w agar flat dish and added to 5m l of T B medium supplemented with 200ug/m l of kanamycin and 10% T B s u p p l e m e n t . 19. Incubate at 37°C for 2~4 h with shaking at the usual speed. It is recommended to start culturing the bacteria when the library is injected into the animal. 20- Dilute a portion of the bacterial solution with T B at I : 10 and measure the OD600. When the OD600 was in the range of 0.16~0.20, reduce the speed to shiver the bottle. 2 0 , reduce the rotation speed to shudder the shear bottle force. Use within 30 m i n . Recovery of phage from cell pellets 21-Add 1500ul of K 9 1 kan bacteriophage solution to the phage cell pellet and gently resuspend the pellet, be complete. Incubate at 37°C for 30 m i n , vortex or up and down the sample for l O m i n . 22. Transfer the bacteria to a 500 ml cell culture flask, add IOOml of pre-warmed tetracycline containing 0.2 ug/ml and incubate at 37°C for 30 min. 23- Remove 10~IOOOul of the suspension containing 40 ug/m l of tetracycline to calculate the phage transduction units in each tissue. 24-Adjust the concentration of tetracycline in the remaining bacteriophage suspension to 20ug/m l , and incubate overnight at 37°C on a shaker. 25. Centrifuge the bacterial solution at 800 r/m i n for 15 m i n and pour the supernatant into a clean test tube. When decanting, do not pour the cell pellet into the upper part of the test tube. 26. Add 1.5 ml of PEG/NaCl to every 10 ml of supernatant, shake, and incubate on ice for 111 ml. Samples can also be incubated at 4°C overnight. 27. Centrifuge at 8000r/m i n for 20m i n at 4°C and gently decant the supernatant. 28. Place the centrifuge tube in the rotor with the side containing the pellet facing the outside of the rotor. Centrifuge at 8000r/min for 5 min to remove all PEG and further concentrate the precipitate. 29. Quickly aspirate the supernatant using a vacuum pipette or a 200M pipette. 30-Add T B S to the phage precipitate (2 0 0 ~ 400M, depending mainly on the amount) by shaking for l O m i n. Avoid that pipette blowing bubbles will cause re-suspension of the phage precipitate to the detriment of the phage and the peptide(s) being demonstrated. 3 1 . Transfer the solution to an Eppendorf tube and centrifuge at 14 o o o r m i n to remove bacterial fragments. 32. Carefully transfer the supernatant (phage solution) to a new Eppendorf tube, taking care not to touch the precipitate. 33- Recover the phage solution by titration (see Scheme 2 ). 34. Repeat the phage screening (steps 1 to 32), and in each successive round of screening, the concentration of recovered phage used For more product details, please visit Aladdin Scientific website.
K 91 ka n Growth of Bacteria
Incubate the culture for at least 12 h, but not more than 16 h. Recovery of phage from the culture broth
