Reverse transcription virus vector assay
Reverse transcription virus vector assay
Reverse transcription viruses are well suited to be used as gene introduction tools because of their specific life cycle and ability to integrate efficiently into the DNA of the target cell. Two regulatory regions, also known as long terminal repeats (LTRs), flank the genome of retroviruses, function as promoters and enhancers and are also required for integration into host genes.
Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Reverse transcription virus vector assay Move Transient Transfection Production Carriers Materials Reagents EKLO medium: DMEM medium with 10 % fetal bovine serum (DMEM-10), 2 mmol/L L-glutamate, 100U/ml penicillin, lOOjug/ml streptomycin. HEK 293T cells (ATCC, CRL-11268) HEPES Buffer Solution (HBS): Mix lOOpl of 500 mmol/L HEPES-NaOH (pH 7.1), 125ul of 2.0 Omol/L NaCl, 10ul of 150 mmol/L Na2 HPO4-NaH2PO4 (pH 7.0), and add H2O to lml. freshly prepared. Phosphate Buffer Solution (PBS) Plasmid Packaging plasmids containing virulence >0/ and ewt; genes Vector plasmids Preparation for extraction of endotoxin-free plasmids (e.g., with the Q IA G EN Endotoxin-Free Purification Kit) and determination of plasmid D N A content. Equipment Biological Safety Cabinets CO2 Incubator Aseptic Tip Injection Filter (0.45um pore size) Tissue Culture Centrifuge Tissue culture flask (75 cm2) Methods 1.Preparation HEK 293T single cell suspension was inoculated in 75 cm2 tissue culture flasks at a density of 5 X IO6 and incubated overnight. 2. Remove the flask and discard the medium, add 12 ml of fresh D>10 medium and continue incubation. 3. All reagents should be filtered through 0.22um pore size sterile membrane before use. Dilute the plasmid with water to a total volume of 876ul, add 124ul of 2.Omol/L CaCl2, mix gently, and then add this DNA suspension (Iml) dropwise to Im l of HBS, and let the cloudy solution form, leave it at room temperature for 30 min, mix gently, and then add I.5 ml of DNA/HBS suspension to the culture flask and incubate overnight. 4. Discard the medium in the culture flask, rinse with 5 ml of PBS, add 12 ml of fresh medium to the flask and continue to incubate for 20~24 hours. 5. Remove the medium containing the carriers from the culture flask, filter through a 0.45um filter to remove residual cells, divide as needed, and store at 70°C. reagents D>10 medium: DMEM with 10 % fetal bovine serum (DMEM-10) G418 Select histidinol or hygromycin B according to the appropriate labeling of the packaging cells. PolybreneinPBS (4 mg/ml; 1000X), filtered to remove bacteria (SigmaAldrich). Precipitation buffer Mix 100/ul of 500 mmol/L HEPE&NaOH (pH 7.1), 125/xl of 2. Omol/L NaCl, IOfil of 150 mmol/L NaH2 HPO4-NaH2PO4 (p H 7.0), add water to a total volume of lm l. Prepare fresh. Recipient cells (e.g. NIH-3T3 or HeLa) Tris-Cl (10 mmol/L , pH 7.5) Trypsin-EDTA Vector Plasmid (must be selectively labeled) Include a vector carrying only the selective marker gene without the target cD N A fragment inserted as a positive control to indicate successful completion of the transfection steps, and then use a plasmid without the retroviral vector as a negative control. Prepare the de-endotoxinized plasmid master mix (e.g., with the Q IA G EN Endotoxin-Free Purification Kit) and determine the plasmid D N A content. Equipment Biological Safety Cabinets CO2 Incubator Cloning Ring felt-tip pen Sterile Filter (〇.22um) Glass Petri dish with lid (IOcm) Sterile tip Silicone resin (or alternative) Tissue culture dishes (6 cm and 10 cm) Centrifuge tubes (12 cmX75 cm Falcon2054 or alternative) Tweezers Methods 1. Day 1: PE501 packed cells were inoculated at a cell density of 5X 105 and cultured overnight in 6 cm dishes. 2. Day 2: Replace PE501 with 4 ml of fresh medium and transfect the cells with plasmid DNA in calcium phosphate programmed vector as follows. All reagents were filtered with 0.2 2um sterile filter membrane to remove bacteria before use. a. For each plasmid sample, prepare a DNA-CaCl2 solution and mix as follows. 10ug Vector DNA (in 10 mmol/L Tris-Cl pH 7. 5 solution) Add water to 200ul b. Prepare fresh Precipitation Buffer. Using a clean 12 mmX75 mm polyethylene tube, dropwise add 200ul of DNA-CaCl2 solution to 200ul of Precipitation Buffer, shaking the tube constantly. The mixed solution will quickly turn a light dark color; it is a mistake if the solution is clear or if there are large clumps of precipitate. c. Allow to stand at room temperature for 300 min. Place the mixed buffer in a cell culture dish, shake the dish to distribute the buffer evenly, and incubate overnight. 3. Day 3: Discard the medium from the transfected PE501 cells and add 4 ml of fresh medium. Inoculate PT67 packaging cells at a density of IO5 cells in 6 cm dishes, and prepare two dishes for each PE501 cell to be transfected. Incubate overnight. 4. Replenish PT67 with medium containing 4ug/ml Polybrene. Remove 3m l of virus-containing medium from the dish of each transfected PE501 cell (leave Im l of medium in place so that the cells do not dry out before trypsinization) and centrifuge the removed medium at 3000 g for 5 min to remove cells and debris. For each dish of transfected PE501 cells, infect one dish of PT6 7 cells with Im l of medium containing the virus, infect another dish of PT6 7 cells with IOfjJ, and return the PT67 cells to the incubator. 5. After trypsinization, PE501 cells were inoculated in 6 cm dishes at a dilution of 1:20 in medium containing 0.75 mg/ml G418 (activation concentration), 4 mmol/L histidinol, or ∼0-4 mg/m l of homomycin B, depending on the selective labeling in the vector. Cells were placed back into the incubator. 6. Day 5: Trypsinized PT67 cells were inoculated in l0 cm dishes at 9:10 and I:100 dilutions. The dishes contained IOml of medium and drug of choice. Infection of PT67 cells with Im l or IOmI virus at 9:10 and 1:10 dilutions results in a 4-logarithmic dilution, which produces the appropriate amount of clones for isolation of the cloned cell line. 7. Day 9: Stain PE501 cells and evaluate clone formation after 5 days as a measure of DNA transfection efficiency. Approximately 1000 clones per mg of plasmid; DNA transfection is very representative. 8. Day 9 to 14: Transfect PT67 cells for 5~IOd until drug-resistant clones are formed and can be seen. 9. Use a cloning loop to isolate clones from cloning plates with a small number of clones. a. Prepare the cloning ring for use: spread a thin layer of silicone resin on the lower part of an IOcm Petri plate, place the ring in the plate until the resin covers the edge ends, and autoclave. b. Separate the clones, place the clones and draw a circle on each cloning plate with a felt-tip pen. c. Aspirate the medium, place the cloning loop on top of the cloning colony and press down with forceps. 10. add one drop of trypsin containing ED TA to each container and observe the digestion of the trypsin. 11. When the cells have aggregated, add medium to each ring (-to one) and suck the medium vigorously into the pipette to remove the cells. 12. After amplification, the Southeni method is used to analyze the clonal bands of an intact vector structure, high vector titer, presence of auxotrophic viruses (see marker analysis on the next page), and expression of the inserted gene. Virus collection and analysis 1. Prepare the virus by replacing the medium with a confluent culture that produces vector cells. After 12 to 24 h, collect the culture medium based on 3OOOg centrifugation for 5 min to remove cells and residual debris. Repeat with the same plate of cells 3 to 4 times at 12 h intervals. The virus containing the medium can be used immediately to infect the recipient cells. Freeze at 70°C for future use. 2. Determination of vector titer a. Inoculate recipient cells (e.g. NIH-3T3 or HeLa) at a cell density of 5X 105 cells/6 cm plate for overnight growth. b. Change the medium with medium containing 4/xg/m l of 1,5-dimethyl-1,5-diazoundecylidene polymethylene bromide. Add various ratios of diluted experimental viruses. Incubate overnight. c. Trypsin digest the cells and dilute with medium containing 0.75 mg/m l G418 (activation concentration) at a ratio of 1:20 to facilitate vector delivery of the genes, 4 mmd/L histaminol to deliver the genes, and 0.4 mg/ml thiamphenicol B to deliver the Z1can genes. The concentration was adjusted according to the cell line and the culture was continued. d. After 5~7d, stain and count the clones. Calculate the viral titer. Marker analysis of helper viruses This analysis detects the ability of viral samples from cells that contain a vector but do not produce one to reverse transcribe the vector. The ability to detect a given helper virus is dependent on whether the helper virus can infect cells. For example, ecotropic helper viruses cannot be detected by human cells. Therefore, we must select cells that are compatible with the helper virus to use (Ref. 22). The analysis is a little slow and cumbersome. However, it is very sensitive and is the most important characterization assay in this paper in terms of retrovirus design and ability to induce vectors. Here the following step 8 is available. 1. In order to make cells contain a vector instead of releasing the vector, infect NIH-3T3 or HeLa cells with an unassisted vector that delivers a selective marker (we use LN vectors that deliver genes), and select the cells for the presence of the gene (for G418 in WO). The virus can be obtained from strains that produce high titers of the vector. 2. Within two weeks, cells are passaged to allow potential helper viruses (which should not be existing) to spread. Analyze cellular vector production using NIH-3T3 cells and virus production using HeLa cells, respectively. 3. Save cells that do not produce vectors for labeling for remediation analysis (method described in step 4). 4. Inoculate non-producing cells (NIH-3T3 or HeLa cells) containing M0 vectors identified in step 3 into 5x105 cells/6 cm plates and grow overnight. 5. Non-producing cells are infected with Im l of experimental virus (3,000 g-centrifuged for 5 m in to remove cells and debris), 3 m l of regular medium, and 1,5-dimethyl-1,5-diazidecanoylmethylene poly(methyl bromide) at a concentration of 1 g/ml. A small number of bidirectional helper virus plasmids [e.g., NIH-3T3 transfected with pAM-MLV (Miller and Buttimore 1986)] were available. 6. Passage of cells within two weeks to allow spread of the helper virus (adjustable to 3 weeks for qualification of clinical material, see Wilsonetal.1997). Handle with care not to cross-contaminate cultures, some of which are initially infected with replicating viruses at very high titers, and trypsinize the cells 2 to 3 times in a week. Reinoculate the cells with a dilution of I : 10 to 1 : 40 so that the cells have a relatively high density to allow the virus to spread easily. 7. After 13d or 20d of passaging of non-produced cells (see step 20), inoculate NIH-3T 3 or HeLa cells (same cell type as the non-produced cells used) used for the first time for the experiment in culture plates at a density of IO5 cells/6 cm plate. Grow overnight. 8. Retrieve the medium from the non-producing cells and infect the NIH-3T3 or HeLa cells used for the initial experiment with a lml sample in the presence of 4ug/ml Polybrene. Centrifuge the medium at 300 吆 for 5 min to remove cellular U and debris. Some drug-resistant live cells can be transported with the medium to give a false positive result. Put new qui back into the incubator. Grow overnight. 9. Replenish newly infected cells with medium containing G418 (activation concentration 0 -75 mg/ml for NIH-3T3 cells and L0 for HeLa cells). After 10.5 d, the cells were stained and the number of clones counted. The presence of clonal colonies indicates that the helper virus remediated the Secretary vector in the experimental samples. Usually, it is very obvious that the positive plate is covered with a layer of resistant clonal colonies. reagents Cell culture medium (normal growth medium that enables cell transduction) Ichthyosperm sulfate or Polybrene Retroviral vector material Experimental Cells Equipment Biological Safety Cabinets CO2 Incubator Aseptic Tip Water bath, pre-set at 37°C Tissue Culture Centrifuge Tissue culture flasks Methods 1- Prepare single cell suspension of experimental cells and count the number of cells per milliliter. Prepare cultures for the transduction group and the mock transduction control group. If the drug of choice for the transduction set is involved, prepare a secondary mock transduction control to serve as a selection control for evaluating the potential of the drug. For adherent cells, 2 12m l of appropriate medium containing 5X 102 cells per 75 cundefined tissue culture vessel was incubated overnight at 37°C in a 5 % CO2 incubator. 2. Rapidly thaw the carriers in a 37°C water bath and prepare dilutions. The time from cell exposure to thawing should be as short as possible. 3. Aspirate and discard the medium from the cells. 4. Transduce as follows. a. Vector transduction: add 4 ml of vector containing medium containing: 10ug/ml to each 75 cm2 5 6 vessel. Fish Sperm Sulfate or 8ug/m l of Polybrene. b. Mock transduction: Add im l l of fresh medium containing 0ug/m l of ichthyosin sulfate or 4ug/m l of Polybrene to the appropriate control culture. The addition of polymeric cations such as sperm protein sulfate or Polybrene is intended to enhance the interaction between the anion-charged cells and the carrier particles, resulting in a more than 10-fold increase in the transduction efficiency. IOOX polycation solutions are very easy to configure. If the polycation is used with primary cells and certain cell strains, it is possible to test an appropriate dosage with minimal toxicity for optimal gene transfection. C. Return the cells to a 37°C, 5% (:02) incubator. Expose the cells to the vector for 4 h. In general, more than 4 h does not significantly enhance the efficiency of cell transfection for cell lines. To enhance gene transfection, it is preferable to repeat the transfection over several consecutive days, provided that the cells have entered the transfection cycle. d. After incubation, aspirate the medium from the container, add fresh medium and place in an incubator for further incubation. 5. Optimal Cell Selection or Cell Sorting: Often the resulting vectors exhibit exceptional transgenic power accompanied by transduction of overall enhanced marker genes. If selection is performed, add the selected marker drug after transduction for 24-48 h. Controls include mock transduction cultures with or without the drug. If molecules are expressed on the surface or fluorescent proteins are expressed, vector-expressing cells can be easily categorized, and most of the transduced cell vectors need to be expressed for 72 h to ensure sufficient time. 6. Stable expression of the integrated transgene can be seen in most cell lines after approximately 72 h of transduction. The cells can be used for analysis or amplification in subsequent uses. reagents Cell growth medium (complete and serum-free) Iscove's modified Dulbecco's medium (IMDM) containing 10 % fetal bovine serum (FBS), glutamine, 20^g/ml gentamicin or X-Vivo medium (Cambrex) containing 1 % human serum albumin and 20/xg/ml gentamicin. Cambrex) Cell separation buffer (Invitrogen) Cell For mouse progenitor stem cells, bone marrow cells were collected from 5-fluorouracil treated mice or negative cells were concentrated using VarioMACS (MiltenyiBiotec). For human progenitor stem cells, they were isolated from bone marrow and peripheral blood using the VarioMACS (Miltenyi Biotec) method. Most primary hematopoietic cells were concentrated by fluorescence-activated cell sorting. Cytokine cocktail (usually used to stimulate cells) Flt3-conjugate-thrombopoietin (TPO) + Stem Cell Factor (SCF) (lOOng/ml per serving) Granulocyte Clone Stimulating Factor (OCSF) + Flt3-conjugate + TPO+SCF (50~100ng/ml per serving) OCSF+ TPO+SCF (100ng/ml per portion) Interleukin-6 (IL-6) (lOOU/ml) +SCF (lOOng/ml each) Phosphate buffer solution (PBS) Retronectin Retronectin is available as a low-pressure lyophilized powder or on pre-coated 35 m m plates (Takara, www. takaramirusbio.com or Cambrex, www. cambrejc com). Retronectin material (Img/ml) was prepared in sterile water without endotoxin. Retroviral vector material Equipment Centrifuge Fluorescence Activated Cell Sorting Instrument (FACS) Microscope Petri dish or multiwell plate Disposable plastic products (for handling non-tissue cultures: BDBiosciences) Polypropylene tubing VarioMACS (Miltenyi Biotec) is used to strengthen most primary hematopoietic cells. Fong Fong 1. 12~48 h before transduction, inoculate 2X 105~4X105 cells per milliliter for transduction (medium containing cytokines). Determine the length of the pre-stimulation period and the number of transduced cell cycles using an experimental method. 2. On the day of transduction, coat the plate with Retronectin as follows. a. Dilute Retronectin with PBS in a polypropylene tube, and then, at 2 to 5ug/cm2, coat the plates without tissue culture (see table below for coating at 2jutg/cm2). For example, to coat a 24-well plate, add 4/^1 of Retronectin to 0.5 ml of PBS so that each well is covered. Blow and mix to add to the wells. Tissue-free plates are used because the hydrophobicity of their surface encourages optimal Retronectin bonding. The size of the plate and the concentration of Retronectin coverage: b. Incubate for 2 h at room temperature or overnight at 4°C in plastic wraps. C . Aspirate Retronectin/PBS solution before use. 3. Determine the approximate number of cells transduced. Transduction with Retronectin at a density of I. OXlO5 ~ 2. OXlO5 cells/dn2 4. Next, pre-stimulate, collect and count the cells. The ratio of medium to retroviral vector supernatant was 1 : 1 and supplemented with a cytokine cocktail. 5. After 4 h , collect the cells without wall attachment and centrifuge. Fresh medium containing cytokines was added, centrifuged again, and the cells were put back into the incubator without wall attachment and incubated overnight. This is required for each transduction cycle. On consecutive days, transduce the cells for 2 to 3 cycles, as long as the cells are not too densely packed keeping the cells on the same Retronectin plate. If cells become dense, collect and replace with a new Retronectin plate. 6. After the final transduction cycle is completed, incubate overnight. 7 After resuspending the cells into tubes, pour PBS (5 ml per IOcm plate; 3 ml per well in a 6-well plate) into the tubes to wash each plate. Add separation buffer (and PBS-sample volume) and place in a fume hood for 2-3 min. For more product details, please visit Aladdin Scientific website.
CaCl2 (2. 0mol/L)
CaCl2 (2. Omol/L)
25ul 2. 0mol/L CaCl2
It is very easy to see the clones under the light by holding up the plates, but be careful not to spill the substrate. We have found that turning off the airflow in the air laminar flow setup is very useful to avoid drying out the placed clone rings.
Our typical isolation is about 100 clones for analysis.
cells produce 1^1 or less virus. passaged over two weeks so that the cells were completely infected] served as a positive control for infection. Other helper viruses are able to replicate in non-producing cells.
Tap the edge of the plate to collect residual cells until no cells remain under the microscope.