Site-specific integration experiments mediated by bacteriophage ΦC31 integrase
Site-specific integration experiments mediated by bacteriophage ΦC31 integrase
Efficient delivery of ΦC31 integrase and donor plasmids to recipient tissues or cells of interest remains the most challenging element of this integrative system. Unlike manipulation of the genome using viral methods, the use of ΦC31 integrase almost always requires an additional method to stimulate DNA uptake by the cell. However, the relative simplicity of the plasmid system means that virtually all proven and feasible methods of introducing exogenous DNA into the cell can be realized with ΦC31 integrase. Indeed, a variety of transfection techniques are sometimes shown to be suitable for this application. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Site-specific integration experiments mediated by bacteriophage ΦC31 integrase Move Site-specific integration using bacteriophage ΦC31 integrase Material Reagents Agarose and Sample Buffer Cells: Permanent biochemical mammalian cell line or primary cells DNA Linking Enzyme and suitable linking buffer Dnase Tissue Kinetics (QIAGEN) or other enzymes used to extract genomic DNA. E. coli DHLO B receptor for electrotransfer Fetal bovine serum F u G E N E 6 Transfection Reagent (R o c h e ) or equivalent G 4 1 8 or other appropriate drug of choice Gentamicin sulphate (G I B C O ) or other suitable drugs for selection in mammalian cells Nutritional base High glucose DMEM (Dulbecco’s modified Eagle’s m e d i u m ) LB (Luria-Bertani) agar plates with selective antibiotics L B Liquid Medium with Selective Antibiotics OPTI-MEMI Medium (Invitrogen) Supplemented DMEM medium: high glucose with 9% FBS and 1% penicillin or streptomycin Plasmids Integrase expression plasmid, p C M V I n t (Groth etal. 2 0 0 0 ) and a suitable control plasmid, pCMV-mlnt (Olivares et al., 2002). A plasmid containing the appropriate expression cassette for the gene to be transferred for a species and cell type of interest. If selection is to be made with a drug, an appropriate mammalian resistance gene should be included. Plasmids include the smallest (34bp ) or full-length (approximately 300bp ) a t t B Sequence Outlaw Ij (Groth et al. 2000) p E G F P -C l Reported Plasmids Qiafilter Plasmid M a x i Kit (Q I A G E N ) or equivalent kit Q I A q u i c k Gel Extraction Kit (Q I A G E N ) or equivalent P C R oligonucleotides as shown in Table 1 Phosphate Buffer Solution (P B S ) Restriction endonucleases and appropriate buffers Membrane insulin (0 . 0 5 % ) /E D T A (lmmol/L) (Invitrogen or equivalent) Instrumentation Centrifuge Electroporator (Bio-Rad) or other bacterial transformation methods Flow cytometer Hematocrit Incubator, preset to 37°C Small centrifuge tubes Tissue Culture Plates (6 wells and 100 mm) Tubes (12X 75 Falcon) Methods Transformation 1 . Construct a donor plasmid containing the locus and gene of interest to be transferred and clone the ΦC31 integrase-like sequence into an expression vector. Note that the integration efficiency does not significantly correlate with the position and orientation of the a t t B. This plasmid construction step requires appropriate enzymes and reagents such as L B agar plates containing selective antibiotics and the Q I A q u i c k GEL Extraction Kit. transform the donor plasmid into electro-transfected receptor cells D H l O B Escherichia coli inoculated with the appropriate amount of L B medium . With Qiafilter Plasmid M a x i K i t may be possible to obtain D N A of sufficient purity for subsequent experiments. 2. 24 h before transfection, the adherent mammalian cells were harvested with 0.05% trypsin 1 mmol/L EDTA. The cells were centrifuged at 1200r/min in a tabletop centrifuge for IO min, and the precipitate was resuspended in an appropriate volume of PBS and counted with a hemocytometer. Four wells were laid out in a 6-well plate, seeded at 30 % and incubated overnight at 37 °C with culture conditions suitable for this cell type. 3. Take 100ul of O p t i - M E M I each into 4 small centrifuge tubes, add 3ul of room temperature F u G E N E 6 to each tube, mix gently, and incubate for 5min at room temperature. 4.Add D N A to each tube with . Tube 1: no DNA Tube 2: I f x g p E G F P -C l Report Plasmid Tube 3: 900 ng p C M V I n t and 100 ng transgenic plasmid Tube 4: 900ng p C M V m i n t and 100 ng transgenic plasmid Incubate for 25min at room temperature with gentle mixing. 5 . Remove the medium from each well and add 2 ml of DME medium to each well. Then add 100ul of OPTI-MEMI liposomal DNA complex to each well drop by drop and incubate at 37℃ overnight. 6• 24 h after transfection, the transfection efficiency was detected by flow cytometry as follows: a. Harvest cells from no D N A and lM g p E G F P -C l wells with 1 ml of 0.05% trypsin 1 mmol/L of E D T A . b. Collect cells by centrifugation at 1200 r/m in for 10 min using a tabletop centrifuge. c. Resuspend the precipitate into 200ul of PBS and transfer to a 12X 75 Falcon tube for flow cytometric analysis of G F P expression. In general, transfection efficiencies of more than 15 % are sufficient. In order to establish stable transfection, the following optional operations can be performed. 7. Trypsinize and re-lay two wells of cells into three 100 m m dishes and replenish with DMEM medium, inoculating the 100 mm dishes with 1/3 of the volume of the initial well for each well, and 1/10 and 1/30 for the other two, respectively. The gradient of plate spreading reduces the effect of transfection efficiency on the ability to discriminate immediate resistant clones with selection over time. 8. 24 h after plate spreading, aspirate the medium and replenish with DMEM containing 1.25 mg/m l G 418 (m/V), or other appropriate drug concentration. 9. Replace the selection medium every 4 days, taking care not to lose the primary clones. Unique clones should be readily identified during the subsequent 14d drug selection. Alternative Methods Stabilization of transfected clusters Immediately following the previous step 6, use the following method: 1. Trypsin digest the cells obtained from both wells after the flow cytometric assay. Collect the cells by centrifugation at 1200 r/min with a tabletop centrifuge for 10 m i n and resuspend the cell precipitate into supplemented D M E N medium. 2. Spread the entire culture of each well separately onto a 100-cell culture plate. Allow the cells to grow to a certain density and passivate as usual. 3. After a few generations of passaging, detect the expression of the transferred genes by appropriate methods. Cells initially transfected with ΦC31 integrase and the transgenic slave plasmid should have several times higher expression of the transferred gene than the control (Thyagarajan and Calos 2005). For more product details, please visit Aladdin Scientific website.
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The difference between pCM Vm lnt and pCM VInt is mainly the inactivation of the product it encodes by mutation of the serine at the catalytic site Φc31 integrase