Successful gene transfer requires efficient transport and long-term presence of exogenous genes in order to maintain physiologically relevant and appropriately regulated expression of therapeutic genes within the recipient's sacrificial cells. The optimal gene transfer platform should have unlimited payload_ force and not integrate with the recipient genome. Moreover, it should form autonomous replicative elements within the recipient cell that regulate gene expression over time. Although some potentially viable methods have been developed, specific, cloned and functional reassembly of chromosomal elements into human artificial chromosomes (H A C ) may still be required to fulfill the above requirements. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
α-Guardian BAC Transfection of H T 1080 Cells Move α-Guardian BAC Transfection of H T 1080 Cells Material reagents Endotoxin-free BAC DNA was extracted using a QIAGEN-tip 500 column, and the BAC DNA was resuspended in endotoxin-removed water to a concentration of 0.5~I.Oug/ul. Fetal bovine serum (FBS; H yclone) was used to extract endotoxin-free BAC DNA. F u G E N E -6 (R oche) Commercially available D N A transfer systems, including transfection reagents and electroporation devices, have been evaluated on the basis of transfection efficiencies with small message plasmids (< 5 kb). There are almost no data on the efficiency of transferring high molecular weight O l O O k b ) D N A . In our experiments, platforms optimized on the basis of small DNAs do not necessarily deliver large molecular BACs well. We tested most of the well-known commercial transfection reagents available; in our experimental conditions, the Fugene-6 reagent (Roche) was the most efficient in delivering large molecules of DNA and was directly usable and less toxic. We also experimented with Lipofectamine 2000 (Invitr0 gen) but found it cumbersome, especially when dealing with multiple transfections. G 418 (50m g /m l ; Mediatech) H T 1 0 8 0 Fibrosarcoma Cell Line The culture medium for HT 1080 was aM E M containing 10 % FBS supplemented with 50ug/m l of Gentamicin (GIBCO). Basic medium, Alpha (aM E M ), supplemented with L-glutamic acid (e.g. Cellgro or G I B C 0 ). azurin (l0 m g/m l; Invitrogen ) Instrumentation Cloning Ring Standard tissue culture equipment, including I-O-meter dishes and T-25 flasks Methods α-Satellite B A C Transfection of HT1080 Cells 1 . The day before transfection, spread IO O m m tissue culture dishes with sufficient H T 1 0 8 0 cells to enable coverage of 50 % to 75 % the next day. At least two petri dishes are required for each transfected D N A . 2- Add IOOfjJ serum-free c t M E M and 4 to 6ug D N A in sterilized I. 5m l centrifuge tubes, one tube per dish. Add 15 to 20fJ F u G E N E -6 reagent slowly, flicking the walls of the tube to mix. Carefully observe the solution for the presence of a white precipitate. If present, break it up slowly with a wide bore pipette tip. Under our experimental conditions, transfection of large BACs is most effective when FuGENE-6 : DNA (volume : mass) is 3 : 2, but each laboratory should optimize this ratio. It should be noted with caution that while superhelical macromolecular mass BAC is resistant to the shear destructive force 彳 I, linear macromolecular mass D N A is very sensitive. 3- Incubate the Fugene/DNA mixture at room temperature for 30 to 45 m in, flicking the wall of the tube to mix. Make sure that no precipitate is formed. 4. Gently add the Fugene/DNA mixture to HT 1080 cells with a wide-bore pipette tip, and shake the dish continuously for even distribution. 5. Incubate at 37°C overnight 6. The next day, aspirate the culture solution from the petri dish and add a suitable selection medium (e.g. medium containing 3 ug/m l puromycin or 600 ug/ml G 418). G418 is also known as neomycin or genetidn antibiotic and should not be confused with gentamycin. 7. After clones are formed, pick at least 25-30 clones for further analysis. Transfer individual clones to T-25 flasks using a cloning ring. Replace with new selection medium every 3 to 5 d. Formation of visible clones takes 10 to 10 days. Formation of clones visible to the naked eye takes 10 to 14 d. When transferring individual clones, inoculate another T-25 or T-27 plate and freeze for long-term storage. reagents C E P -17 cr Guardian probe (Vysis) or equivalent non-commercialized probe Colcemid solution (lOjug/ml; KARYO M A X , GIBCO ) Ethanol (70%, 80% and 100%; store at 20°C) Methylbrewamine (ChemiconInternational) H 2O , Milli-Q process (extra pure) H T 1 0 8 0 Fibrosarcoma cells, transfected with B A C D N A and grown into monoclones in T-25 flasks (see Scheme 1) Hybrisol VII (Qbiogene) KCl (0.075m ol/L ) 5.59 g of KCl was dissolved in I L of double distilled water (d d H z O ) to form a hypotonic solution. The solution was filtered through a 0.22 Mm membrane for sterilization. Preheat to 37 °C before use. Methanol (100%). Methanol/acetic acid solution (3 :1 v/v) Addition of D A P I ( V ectashield, V ector Laboratories) M ounting culture solution (anti-color fading) Phosphate Buffer Solution (PBS) 20 X S S C Dissolve 175. 3 g NaCl and 88_ 2 g sodium citrate in 800 m l of water, adjust p H with 14 mol/L H C 1 to 7. 0. Add water to I L d and autoclave well. Dilute before use. Insulin (0.05 % solution) 0.5 % solution) Instrumentation B e n c h to p type microscope equipped with a phase contrast lens Medical centrifuge Copl i n wide mouth flasks for cell culture Lid tabs (#1, various sizes; Fisherfinest, FisherScientific) Fluorescent Microscope Humidity regulator Microscope slide (Fisher Scientific 12-550-43) Pasteur Pipette (9in, glass; Fisher 13-678-20D) Standard Tissue Culture Unit, including 15 ml tubes and T-2 5 flasks Water bath, preheated to 37 °C Methods Preparation of clonal cell lines for chromosome spreading 1. When the T-25 flasks of H T 1080 clones (see Option 1) are nearly full, add I6. SfJcolcemid solution to each flask. Incubate at 37°C for 40-60 min. observe for round dividing cells. 2 . Pre-warm 50 ml of 0.075mol/L K C 1 in a water bath at 37°C. Label a 15m l round tube for each cell line. 3 . Transfer surface cells to round tubes. Wash the flask with P B S to aspirate excess liquid. 4 . Add I m l of insulin solution to each T - 2 5 flask. Incubate at 37°C until the cells separate. Re-add surface cells to the flasks, blow vigorously to suspend the cells, and transfer the suspended cells to round tubes. 5 . Centrifuge the round tube for 5~lOmin at room temperature with a medical centrifuge at 1500~2000r/m in. 6 - Aspirate the water from the cell deposits. Add io m l ○. in each tube while shaking. Add io m l ○ 75mol/L K C 1 to each tube while shaking. Incubate for 12m in at room temperature with the tissue culture lid on. Centrifuge according to the conditions in step 5. Under our experimental conditions, the optimal time for incubating H T 1 0 80 is I 2m in . Some optimization can also be done. 7 - Aspirate the water from the cell deposits. Flick the tube wall to resuspend the cells to avoid clumping. 8. Fix the cells by adding lm l of a 3:l (V/V) methanol/acetic acid solution dropwise to 5 ml. Add an additional 4 ml of methanol/acetic acid to 5 ml. Invert the round tube. 9. Centrifuge according to the conditions in step 5. The cellular precipitates can be stored in a 20°C methanol/acetic acid solution until the chromosomes have spread. Preparing Chromosome Diffusion for F IS H High-quality chromosome spreading for F IS H is known to be susceptible to laboratory environmental factors and operator experience. Many aspects of this process can be described as more of an "art" and different laboratories may apply different protocols. The following experimental protocols are only a preliminary framework and can be adapted and optimized by each laboratory according to its own environment. Chromosome 'landing' experiments are performed in a small, airtight room with controlled humidity. For example, 2 to 3 water baths and humidity regulators can be turned on a few hours before the landing step. 10. Remove the fixed cells from the room at 20°C (see step 9). Centrifuge the fixed cells at room temperature with a medical centrifuge at 1000~2000r/min for 5~IOmin to deposit the cells. Aspirate off the excess fixative and flick the walls of the tube to resuspend the cells. 11. Prepare fresh fixative (30 ml methanol + IO ml acetic acid) and place it in the landing chamber. Place several new microscope mounts in a Coplin wide-mouth flask filled with 1 ○○% methanol. 12. Resuspend the cells with a Pasteur glass pipette by adding enough of the fresh fixative to make the solution slightly cloudy, but the pipette can still be seen. 13. Dry a microscope slide by blowing vigorously into the slide at least 3 times to moisten it; a visible haze forms on the landing surface of the slide. Bring the slide to waist level with the glass pipette at shoulder level. Make sure that the cells are in full suspension (no clumping!). Place 1 to 2 drops of cell suspension on the carrier sheet. 14. Blow vigorously on the surface of the carrier sheet at least 3 times to moisten it. Place the carrier on the airflow of the humidity regulator for at least 10 s. Blow on the landing surface at least 3 more times to keep it moist. Dry vertically. If necessary, wipe the back of the carrier sheet with cottonless paper. Do not place the carrier too close to the humidity regulator. Just keep it moist, no droplets should form. 15. After the slide has dried, observe the diffusion with a phase contrast microscope and 20X eyepiece. The chromosomes should be clean, dark and homogeneous with good spreading, but it is clear that they were formed by the lysis of a single cell. If the chromosomes are not in a good state of spreading, try adding acetic acid directly to the fixative and repeat. Alternatively, try dropping from a higher height, adjusting the methanol:acetic acid ratio to 1:1, and raising or lowering the ambient humidity. 16. Add IOml of fresh fixative to the residual cells and centrifuge at 1000-200 r/min in a medical centrifuge for 5~l0 min. Cells can be stored in a 20°C fixative before use. 17- Dry the slides thoroughly overnight on a bench or in a vacuum cabinet. The slides should be stored in a desiccator for 1 to 14 d before hybridization. Alpha-satellite probe hybridization for chromosome spreading 18- Incubate diffusion-dried slides in 2X S S C for 30 to 90 min in a 37°C water bath. 19. Pre-cooled 70% ethanol (stored at 20°C) is packed into Copliii wide-mouth vials and the following series of ethanol washes are performed at 120°C to dehydrate the chromosomes by diffusion before taking to the water bath. a. 70 % ethanol wash for 2 min. b.80 % ethanol wash for 2 min. c. 100% ethanol wash for 2 min. d- Remove the slides from the ethanol solution, one at a time. Blot the back with cottonless paper. e. Place on a bench to dry for lOmin. Wash slides at 20.72°C in 7 0 % formamide/2X S S C (P H 7.0) solution for 2 m i n denatured samples. Repeat the ethanol washing process from step 1 9 . Pre-cooled 7 0 % ethanol was loaded into Coplin wide-mouth vials before getting to the 72°C water bath. Place on a bench to dry I O m i n or overnight. 21. Add 0 -5/nl Vysis C EP-17 probe (or equivalent non-commercial probe) to 20ul of Hybrisol VII and prepare one tube for each slide to be hybridized. Denature at 72°C for 5 miri and place on ice. 22. Add the denatured probe directly to each slide and cover with a coverslip. Do not close with playdough or nail polish. Incubate overnight in a moist box (e.g., a plastic box in which wet paper handkerchiefs have been placed; use two I O m l pipettes as holders for the slides). 23. The next day, wash the slides as follows. Remove the coverslips in the first washing step. a. Wash the slides at 42°C in 6 5 % formamide/2X S S C (p H 7 . 0 ) for 8 min (more stringent washing for higher levels of cr satellite sequences) b- Repeat the wash with fresh 6 5 % formamide/2X S S C (p H 7.0). c. Wash at 37°C in 2X S S C for 8m i n . d- Rinse the slide with a Coplin light-topped vial filled with Milli-Q treated water. Shake off excess water and blot the back with cottonless paper. 24- Add 20 to 40 ul of DAPI/anti-decolor mounting culture solution (Vectashield) to the moist slide. Gently place a #1 22X 50 coverslip on each slide using a P2 0 pipette tip. Gently blot excess Mounting Medium from the surface of the coverslip with cottonless paper. Seal the edges of the coverslips with nail polish. Place the slides in a dark slide box at room temperature for at least 2 h prior to fluorescence microscopy. Store at 4°C for long term storage. reagents Colcemid solution (lOfxg/m l ; K a r y o M A X , G I B C O ) Methanol/acetic acid solution (3:I V/V) Add Munting culture solution from DAPI (V ectashield, V ector Laboratory). Phosphate Buffer Solution (PB S) PBS/0. 2% Triton X-100 - Anti (anti-CENP-A mouse monoclonal antibody; S tressg en B io tech n o lo gies) Secondary antibodies (e.g., anti-rat IgG/rhodamine or rhodamine; Jackson Immunoresearch) 20 X SSC Dissolve 175.3 g of NACL and 88.2 g of sodium citrate in 800 ml of water, adjust pH to 7.0 with 14 mol/L H C 1. Add water to 1 L. Dissolve thoroughly and autoclave. Dilute before use. Apparatus Benchetto microscope with phase contrast tip Medical centrifuge Fluorescent Microscope Shandon C ytospin 4 cytocentrifuge (T herm o E lectro n ) Shandon D ouble C ytofunnels (T herm o E lectro n 5991039) Shandon D ouble C ytoslide, encapsulated (T herm o E lectron 5991055) Standard tissue culture device, consisting of 15 ml round tubes and T-25 flasks 1- Culture the selected clonal cell lines in T-25 culture flasks. After 80% to 90% full growth, add 25ul of Clominde solution to each bottle. Incubate at 37°C for about 45min. Observe the production of round, dividing cells under a microscope. 2- After C olcem id incubation, transfer most of the culture solution from each culture flask to a 15 m l round tube. Tap the T-25 culture flasks vigorously on the bench 15 to 20 times. Rinse the newly detached cells with the culture solution in the round tubes and transfer the culture solution (and cells) back into the 15 ml round tubes. 3 . Centrifuge the cells at 200 rpm in a medical centrifuge at 4°C for lO m in. Aspirate the culture solution and flick the walls of the tube to keep the cells suspended in the residual culture solution. 4 . Add I m l of preheated (37°C) 0.075mol/L K C l dropwise to each tube of cells while stirring. Add an additional 4 ml of pre-warmed 0-075mol/L KCl to each tube. Place the remaining 0-075mol/L KCl on ice and incubate at 37°C for 30 minutes. 5-Assembly of labeled Shandon D ouble C y tofunnels. Cells were deposited using the corresponding encapsulated microscopic mounts designed for C y to sp in with two spots on each mount. 6 . Centrifuge the cells at room temperature with a medical centrifuge l ○○ r/m i n for 5m i n . Gently pour out 0.075mol/L K C l . Resuspend the cells with 3m l of pre-cooled ○ - ○ 75m o l / L K C l on ice. .... Blow vigorously with a transfer pipette to ensure total suspension. 7- Add 250~300ul of cell suspension per Cytofunnel. Centrifuge at 200r/m i n for lOmin at room temperature. 8 . Remove the slides from the CY to SP in and dry for lO m in. Do not dry completely. Incubate the slide in PBS/0-2 % Trinitron X-100 for 6 m in at room temperature. 9. Observe chromosome spreading with a phase contrast microscope and 20 X eyepiece. 10. Fix the cells with 4 % formaldehyde/P B S /0. 2 % Triton X-100 for lOmin at room temperature. 11- Wash the loaded slices with P B S for 5m i n , and immediately contribute with a primary antibody (anti-C E N P -A mouse monoclonal antibody, diluted with P B S). Cover each slide with a cover slip (do not close) and incubate overnight at 37°C in a humid cabinet. The antibody can be diluted at a concentration of 1/50 to 1/500. 12. Wash the mounts twice with PBS for 5 m i n each time. 13. Add IOOm I of secondary antibody (e.g. anti-mouse IgG covalently bound to FITC/TRITC) diluted with PBS l:200 to each slide. Cover with an unsealed cover slip and incubate for 1 h at 37°C in a humidified cabinet. 14-Wash the mounts twice with PBS for 5 m i n each time. 15-Fix the sample with 4 % formaldehyde/P B S /0. 2 % Triton X-100 for IOmin at room temperature (see step 100). 16. Wash the slide twice with water, 5 m i n each time. 17. Fix the sample with methanol/acetic acid solution (3:IVAO) for 15 m i n at room temperature. Dry the slide in the dark for 5 m i n at room temperature. 18. Denature the sample in 70% formamide/2X SSC at 85°C for 11 mIn. 19. Perform the following series of ethanol washes at 20°C to dehydrate the samples. a. 70 % ethanol wash for 2 m i n . b. 80% ethanol wash for 2 m i n . c. 100% ethanol wash for 2 m i n . d. Dry the slide in the dark at room temperature. 20. Incubate the F IS H probe at 85°C for 5 m i n denaturation when the 80 % ethanol dehydration step in Step 19 begins (see Scheme 4). Store at 37°C until use. 21. Add 7 ~IOul of probe to each cell circle and cover with a 22m m X 22m m square cover piece (do not close). Incubate overnight at 37°C in a humid cabinet (e.g., a plastic box with wet paper handkerchiefs; use two IO m l pipettes as slide holders). 22.42°C Wash the slide twice with 50 % formamide/2X SSC for 10 m i n each time. Then wash the slides in 2X SSC for IOmin0 at 42°C. 23. Dry the back of each slide. Add 3 drops of 8ul Vectashield to each slide and cover with a #1 24X 50 cover sheet. Seal the edges of the cover sheet with nail polish. Store at 4°C until visualized by fluorescence microscopy. carve on trial basis Prepare electrophoretic grade BAC carrier backbone D N A solutions at concentrations ranging from ○.2 ug/ul to Iug/ul with Tris-EDTA (lOmmol/L T ris, lm m ol/L EDTA, pH 8. 5 ) buffer. C O T-1 D N A (Invitrogen) dNTP mixing solution (〇.lmmol/L) Mix 10/J of 0.3 mmol/L dA TP, 0.3 mmol/L dCTP, and 0.3 mmol/L dGTP. d T T P (0. lm m o l/L ) Add IOul of 0. 3 mmol/L dTTP to 20ul of nuclease-free water. The dU T P (0-2 mmol/L ; SpectrumGreen, SpectrumOrange, or SpectrumRed; Vysis) Add 10M100 - 3 mmol/L dU TP to 40ul of nuclease-free water. Ethanol (1 0 0 % ) H2O (no nuclease) Human Placenta DNA (Sigma-Aldrich) H ybrisol V II (Q bigene) nick T ran slatio n K it (V ysis) Sodium acetate (3m ol/L, p H 5.2) Instrumentation Standardized equipment for agarose gel electroswimming, including molecular mass specification Methods Labeling probes 1. Place a centrifuge tube on ice to cool. 2. Add the following components in the order listed below. Vortex with slight centrifugation before adding the enzyme. 17.5 ~ x ul NER water X ul DNA extracted (I pg) 2.5 ul d U T P (0.2 m m o l/L S pectrum G reen, S pectrum 5ul d T T P (0. lm m o l/L ) IOul d N T P mixing solution 5ul IO X n ick tran slatio n buffer 10ul nick tran slatio n enzyme Total volume 50ul 3. Slightly centrifuge the vortex tube. Incubate at 15°C for 8~16 h. 4- Terminate the reaction by heating I O m i n a 70°C water bath. Cool on ice. As the amount of enzyme and the incubation time increase, the number of small probe fragments increases. To obtain small probe fragments, apply the following conditions (in order of decreasing fragment size): 5ul of enzyme mixture + 8 h incubation, 5ul of enzyme mixture + I6 h incubation, IOftI of enzyme mixture + 8 h incubation, and IOul of enzyme mixture + 16 h incubation. The amount of nuclease-free water was adjusted to control the reaction time. 5- Spot sample 10ul Check the size of the probes by electrophoresis on a 2 % agarose gel. Most of the probes should be around 3,000 bp. Precipitate the probes 6. Add 5 ul (approximately I O O n g of probe) of the incision pan reaction mix to the centrifuge tube. Add 1 ug COT-1 DNA, 2ug Human Placenta DNA, and 4ul of purified water. Add 1.2ul (0.1 volume) of 3m o l/L sodium acetate (p H 5.2) and add 30ul (2.5 volume) of 100% ethanol to precipitate the DNA. Lightly vortex on dry ice for 15 m i n . Centrifuge DNA at 12,000r/min at 7.4°C for 30min. 8. Remove the supernatant and dry the precipitate under vacuum at room temperature for 10 to 15 millimeters. The precipitated probe can be stored in the as-deposited state at -20°C. 9. Resuspend the precipitate in IOul of Hybrisol VII. Before use, denature by heating for 5 m i n in a water bath at 85°C (see Scheme 3, step 20). Resuspended probes can be stored at 4°C for less than 2 weeks or at 20°C for an unknown length of time. For more product details, please visit Aladdin Scientific website.
BAC D N A
acetic acid (CH3COOH)
Preheat to 37°C prior to
Methods
Incubate the cells at 37°C for 12mn.
Chromosomes should be brightly colored with little difference from the background, so they are somewhat difficult to see. If a few diffusions can be seen, it can be concluded that there are many unseen diffusions present. If the diffusion is dense, use more ○. Repeat the Cytospin procedure with more 0.75 mol/L K C l diluted cells.
BAC D N A Stencil
O range or S pectrum R ed)
Adjust the amount of nuclease-free water to control the total reaction volume at 50ul.