Lentiviral vector is a new type of transgenic vector developed in recent years. Lentiviruses belong to the subfamily of retroviruses. After structural modification, lentiviral vectors do not proliferate in host cells and do not lead to the death of host cells, and the infected animal cells can be passed on to normal successive generations, and their greatest advantage lies in their ability to infect static cells as well as dividing cells, and their infection rate is extremely high.
Operation method
Preparation of transgenic animals by infection with recombinant retroviral (lentiviral) vectors
Materials and Instruments
Equipment: Move The basic process of preparing transgenic animals by infection with recombinant retroviral (lentiviral) vectors can be divided into the following steps: Recombinant lentiviral vectors are generally derived from the modification of natural lentiviral genomes (e.g. HIV-1 genome). Commonly used lentiviral vectors have completely eliminated all structural protein genes from the original lentiviral genome, retained the DNA cis-acting Flap-1, which can increase the integration rate of the original virus and the viral titer, and added the post-transcriptional regulator of gene expression (WPRE), which is derived from the Wooduck virus genome and can increase the expression level of the transgene; furthermore, the U3 in the 3＇LTR of the viral vector has been deleted. In addition, part of the U3 region in the 3' LTR of the viral vector was deleted, resulting in the automatic loss of self-inactivation of the LTR sequence after the viral genome was reverse transcribed into provirus and integrated into the host cell genome, so that exogenous tissue- or organ-specific promoters could be inserted to achieve tissue- or organ-specific expression of the target genes. 1. Inoculate 293FT cells in T150 bottles and culture them to 50% fusion, using the following medium: high sugar DMEM + 10% FBS, 1% Glutamax + 1% (penicillin-streptomycin). (2) Isolate the 293FT cells from the T150 bottle and distribute the cell suspension into 12 10 cm cell culture dishes (about 1x108~109 cells/dish), each containing 25 mL of the same high-sugar DMEM medium as in the previous step. . . Examine the inoculated cells under a microscope, and when the cells show 60% to 80% confluence and good wall adherence, they are ready to be used for transfection. 2. Add 25 μl of 25 mmol/L chloroquine to each culture dish to achieve a final concentration of 25 μmol/L. 3. 3. In a 5 ml centrifuge tube, mix 72 μg of plasmid pMD2.G, 180 μg of plasmid psPAX2, and 240 μg of plasmid FUCW in sterile deionized water, and dissolve to a final volume of 13.14 mL, followed by the addition of 1.8 mL of 2 mol/LCaCl2. 4. 4. Mix the solution thoroughly and aliquot 1.25 mL of the mixture into 12 sterile 5 mL conical centrifuge tubes. 5. 5. Using a 5 mL pipette, add 1.25 mL of 2xHBS (280 mM NaCl, 10 mM KCI, 1.5 mM Na2HPO4,12 mM sucrose, 50 mM Hepes, pH 7.00-7.45) dropwise to the mixture dispensed in the previous step and vortex it gently (to ensure sufficient mixing in the tube) to obtain the DNA-calcium phosphate complexes (Note.): Proper preparation and preservation of 2xHBS is very important) 6. Allow 1-2 minutes for calcium phosphate precipitation to form, then gently drop 2.5 mL of the mixture from step 5 into a 10 cm dish of 293 FT cells. 7. Repeat steps 5 and 6, and add the DNA-calcium phosphate mixture dropwise to the remaining 11 10 cm dishes containing 293FT cells. 8. Transfer the 12 dishes to a cell culture incubator and incubate overnight (16-18 hours) in 5% CO2 at 37 °C (note: make sure that the dishes are balanced in stacks of up to 6 dishes each). 9. Remove the medium by aspiration and add 17 mL of fresh high-sugar DMEM (containing 10% FBS, 1% glutamine and 1% penicillin-streptomycin) and continue incubation for 48 hours at 5% CO2, 37 ℃. . Observe the transfected cells under a microscope. The syncytia of the fused cells should begin to appear and most cells should still have the ability to adhere. If there is a reporter gene encoding a fluorescent protein (e.g., EGFP), more than 95% of the cells will be seen to fluoresce (Figure 5-1-42). 2. Collect the cell supernatant from the cells in a 50 mL pointed-bottom centrifuge tube and centrifuge the tube at 500 g for 10 minutes at 25 °C to remove cells and large cellular debris, and filter the centrifuged supernatant through a 0.45 μm filter into another clean 50 mL pointed-bottom centrifuge tube. 3. Centrifuge at 20,000 g for 6 hours (or 70,000 g for 2 hours) (to increase titer, 100 mL of supernatant can be centrifuged twice in a row in the same 50 mL tube; discard supernatant and determine location of precipitate. 4. Resuspend the precipitate with 200 ul of HBSS (Invitrogen Cat. No.: 14025-092). Resuspend 100 μl of the precipitate, then rinse the tube with another 100 μl and mix the two suspensions. 5. Take a 1.5 mL tube, add 400 μl of HBSS buffer containing 20% sucrose, then add the virus suspension above the level of HBSS buffer and centrifuge at 50,000 g for 2 hours at 4 ℃. 6. Discard the supernatant. 6. Discard the supernatant, resuspend the precipitate with 70 μl of HBSS, centrifuge at 4 ℃ and 12000 g for 10-30 seconds, separate the supernatant and store at -80 ℃. . Inoculate 5x10 293 FT cells per well in a 6-well plate. 2. Add 2 μl, 5 μl, 10 μl, 100 μl, 200 μl, 500 μl of virus solution diluted 1000-fold into each well of the 6-well plate inoculated with 293 FT cells (the final number of times of dilution should be adjusted at any time according to the results of the flow cytometer in order to get the best linear results and calculate the viral titer with relative accuracy). 3. After 48 hours of infection, replace the medium with 2 mL of fresh medium and continue to incubate the cells for 48 hours. 4. Remove the medium and wash the cells 2-3 times with 1 mL of PBS, then add 0.5 mL of trypsin-EDTA solution to each well and incubate at 37 ℃ for 2 minutes. 5. 5. Add 1 mL of medium to each well and mix well to terminate the digestion, and transfer the cell suspension from each well to a 5 mL sharp-bottomed test tube and centrifuge at 500 g for 5 minutes at 20 ℃ to obtain the cell precipitate. 6. Discard the medium, resuspend the cells with 2 mL of Hanks' Equilibrium and centrifuge at 500 g for 5 minutes at 20 °C to obtain the cell precipitate; then discard the supernatant and resuspend the cells with 300 μl of Hanks' Equilibrium. 7. The percentage of green fluorescent cells was analyzed by FACS9 flow cytometer. . Acquisition and collection of early embryos from animals. 2．After the embryo extraction, a flat droplet was prepared on a concave slide with 10 μl of M2 culture solution, covered with liquid paraffin, and incubated in a CO2 incubator. 3. Thaw the virus solution before injection and centrifuge at 4 ℃ and 12000 r/min for 1 minute. 4. 4. Use Mierolodder (Eppendorf) to enter from the end of the injection needle, and load a small amount of virus solution after reaching the tip of the needle. 5. Add liquid paraffin into the injection needle with a homemade glass capillary tube, expel the air inside the injection needle, and the gas between the virus liquid and liquid paraffin. 6. The egg holding needle and injection needle were installed in the microscopic operating system of the left and right operating arm, and microinjector connected, adjusted to the appropriate height and angle. 7. Inhale 2/3 length of M2 culture solution with the ovipositor needle, leaving no air bubbles at the front end. 8. Remove the concave slide and fill the microtitre with 50-80 embryos with clear male and female prokaryotic nuclei and place them on the inverted microscope stage. 9. Before inserting the syringe needle into the first embryo, gently touch the tip of the needle to open the tip, expel a small amount of gas from the tip, adjust the pressure inside the syringe needle, and aim the syringe needle at the embryo under the microscope, and if the embryo moves, the tip is opened. 10. Puncture the needle into the zona pellucida of the upper (or lower) half of the embryo from the 3 o'clock direction and withdraw the needle immediately when the perivitelline space becomes larger and the zona pellucida becomes thinner (Fig. 5-1-43a, b), and then use the needle to suck up the next egg for injection. 11. After the embryos in the droplet are injected, transfer them back to the M16 culture droplet for incubation, and then proceed to the next batch of injection. For more product details, please visit Aladdin Scientific website.
①T150 bottle, Petri dish
②Microscope
③Centrifuge
④37 ℃, 5% CO
2
④37℃, 5% CO2 incubator
⑤FACS9 flow cytometric analyzer
⑥Mierolodder (Eppendorf)
⑦ Homemade glass capillary tube
Reagents:
①293 FT cells
② High sugar DMEM + 10% FBS, 1% Glutamax + 1% (Penicillin-Streptomycin)
③chloroquine, Na
2
HPO
4
④M2 culture medium, M16 culture medium
⑤Hepes, HBSS (Invitrogen Cat. No.: 14025-092), PBS
⑥Trypsin-EDTA solution
⑦Hanks equilibrium solution