Knockout technology based on homologous recombination of embryonic stem cells
Knockout technology based on homologous recombination of embryonic stem cells
Homologous recombination is a type of genetic recombination, which refers to the rearrangement of DNA caused by the exchange of nucleic acid sequences between two similar or identical DNA strands. This process involves several steps, including DNA breakage and rejoining. Although homologous recombination often repairs DNA double-strand breaks, it also mediates chromosome exchanges during meiosis, resulting in recombinant DNA. these mutations due to homologous recombination are an effective way for organisms to adapt to changes in their environment. Gene knock-out is a technique to inactivate or deactivate specific genes in an organism through a certain pathway, and is an experimental method to change the genetic information of a cell or an individual in a targeted manner. It is based on embryonic stem cell (ES cell) technology and homologous recombination technology.
Operation method
Knockout technology based on homologous recombination of embryonic stem cells
Materials and Instruments
Equipment: Move The main operational steps of gene knockdown technology based on homologous recombination of embryonic stem cells are briefly described as follows: (1) Extraction of BAC: The extraction of BAC is basically the same as that of ordinary plasmid, the only thing to be noted is that the BAC is larger, so it should be operated gently to avoid destroying its integrity, please refer to Section 4 of Chapter 1 of this article for details. (2) Preparation of electroporated sensory bacteria and electroporation of BAC (preferably freshly extracted products) (1) Inoculate monoclonal EL350/EL250 into 3 mLLB medium and incubate at 32 ℃ 240 r/min for 12~16 hours. (2) On the second day, inoculate 1 mL of overnight bacterial solution into 50 mLLB medium and incubate at 32 ℃ 240 r/min for 2~3 hours until the OD600 is 0.5~0.7. (3) (If only BAC is transferred without homologous recombination, this step is omitted.) Dispense 10 mL of the bacterial solution into 200 mL conical flasks and place them in a 42 ℃ water bath shaker (about 40 r/min) to keep warm for 15 minutes. 4) Place the conical flask on ice for 20 minutes, during which time pre-cool MilliQ water pH 7.4 or 10% glycerol, as well as the rotating beaker and centrifuge tube on ice. 5) Centrifuge at 3500 r/min for 5 min at 4 °C to precipitate the bacteria. 6) Gently resuspend the organisms in 1 mL of ice water, centrifuge at 3500 r/min for 5 min, and pour off the supernatant; repeat this step 3 times. 7) Pour off the supernatant and use the remaining 50 μl to suspend the bacteria. 8）Mix the susceptible bacteria with 100~400 ng of DNA (must be dissolved in MilliQ) and transfer to a pre-cooled 0.1 cm electrotransfer cup and place on ice for transformation. 9) Set up the electrotransfer apparatus, 1.75 kV, 25 μF, 200 Ω, for electrotransformation. (10) After electroshocking, quickly add 500 μl SOC and keep warm at 32 ℃ for 30 minutes; evenly apply the bacterial solution on the corresponding LB agar plate and keep warm at 32 ℃ for 12-20 hours. (3) PCR chimeric primer design and PCR purification: the primers were designed in the pattern of 5'--- homology arm sequence + amplification primer ----3'. The PLoxp primer was used as a template with the pl452 plasmid for the amplification of Loxp-NEO/Kan-Loxp fragments of homology sequences of ～80 bp at each end (60-70 was acceptable, in any case, try to make sure that the total length was not more than a hundred), and the Pfrt primer was used with the pl451 plasmid as a template for amplification of Loxp-NEO/Kan-Loxp fragments of the same sequence. Pfrt was used to amplify FRT-NEO/Kan-FRT fragments of ～80 bp each, using the pl451 plasmid as a template. Note: pl451 and pl452 used as templates need to be digested and linearized, otherwise the plasmids will be easily recovered together during gel recovery. The above PCR products and digested fragments were purified by gel recovery. (4) Extraction of the target fragment from the BAC: The EL350 transfected into the BAC is prepared as an electroreceptor and the linearized vector PL253-5'-3' ARM is inserted into it (100-200 ng/50 μl of the receptor, but not too much, as this may introduce too much uncut plasmid and result in a high background). After electrotransformation, the bacteria were screened by LB plates with Amp+. (5) Insertion of Loxp-NEO/Kan-Loxp fragments: The EL350 containing PL253-fragment was prepared as an electroreceptor, and the purified Loxp-NEO/Kan-Loxp fragments were transferred into it (100-200 ng/50 μl of the receptor). After electrotransformation, LB plates were screened with AmpKan+. (6) Cre-mediated removal of NEO fragments between Loxp sites (1) Inoculate the EL350 clone containing Loxp-NEO/Kan-Loxp plasmid into 3 mL of LB medium (Amp＊Kan+) and incubate at 32 ℃ for 12-16 hours at 240 r/min. (2) On the second day, transfer 1 mL of overnight bacterial solution into 10 mL LB, and incubate at 32℃ for 2~3 hours until OD600=0.5. (3) Add 100 μl of 10% L (+)-arabinose and continue to incubate for 1 hour. (4) Dilute the bacterial solution and apply it on ampicillin and kanamycin LB agar plates respectively, and incubate at 32 ℃ overnight. 5) There should be far more clones on the ampicillin plate than on the kanamycin plate. Pick a few clones on the former to culture and verify by PCR, digestion or sequencing. (7) Insertion of FRT-NEO/Kan-FRT fragments: The plasmid integrating the first Loxp is transferred into EL350 and prepared as an electro-transferred sensory state, and the purified fragment FRT-NEO/Kan-FRT is transferred into it (100-200ng/50 μ1 sensory bacteria). 1. Preparation of feeder cells (1) Take E12.5~E14.5 mouse embryos. Place them in 10 cm Petri dishes with D-hanks. (2) After removing the head and viscera, transfer the remaining parts into the lid of a 10 cm dish and cut them as much as possible with sterilized elbow scissors. (3) Add 3 mL of Trypsin/EDTA. incubate at 37 °C for 10 minutes, shaking constantly by hand. (4) Add 3 mL of 10% FBS-DMEM. 37 ℃ for 10 minutes, shaking constantly by hand. (5) 1000 r/min for 5 minutes. Carefully aspirate the supernatant with a pipette gun, allowing a small amount of liquid to remain. (6) Add 10 mL of 10% FBS-DMEM and resuspend. Spread onto a 10 cm Petri dish. Label as MEFs P1. (7) Incubate at 37 ℃ for two days and pass on 1:3 to 1:5. Labeled as MEFs P2. (8) Incubate at 37 ℃ for two days. Freeze P2 (1 dish and 3 tubes). Label as MEFs P2 (freezing solution: 95% FBS + 5% DMSO). When the cells reach 90% confluence, MEF cell passaging can be performed. Aspirate the medium under negative pressure, add 4 mL of DPBS, aspirate the DPBS, and add 1.5 mL of Trypsin/EDTA. Leave the cells in the cell culture incubator at 37 ℃ for 3 minutes. Add 4 mL of 10% FBS-DMEM to arrest the effect of Trypsin. (9) Transfer the cells to a 50 mL tube and centrifuge at 1000 r/min for 3 minutes. Aspirate the supernatant under negative pressure. Resuspend cells with 30 mL of 10% FBS-DMEM. Spread onto three 10 cm Petri dishes. After the cells have grown to their full size, prepare a feeder layer of cells and aspirate the medium under negative pressure. Add 10 mL of fresh 10% FBS-DMEM and 10 μg/mL mitomycin C. Place the cells in a cell culture incubator at 37 ℃ for 3 hours. (10) Aspirate off the mitomycin C and store at 4 ℃ away from light if it is the first use, or do not keep it if it is the second use. Add 10 mL of DPBS, aspirate off the DPBS and repeat 3 times. (11) Add 1.5 mL Trypsin/EDTA and place in a cell culture incubator at 37°C for 5 minutes. The effect of Trypsin was interrupted by the addition of 4 mL of 10% FBS-DMEM. (12) Transfer the cells to a 50 mL tube and centrifuge at 1000 r/min for 5 minutes. Aspirate the supernatant under negative pressure. 30 mL of 10% FBS-DMEM resuspend the cells and spread them onto 3 10 cm dishes pretreated with 0.1% gelatin and incubate at 37°C. Use within 1 week. 2. ES cell recovery (1) Remove one tube of ES cells from the liquid nitrogen tank and quickly lysed in a 37 ℃ water bath. (2) Transfer ES cells and 2 mL of ES-DMEM to a 15 mL centrifuge tube and centrifuge at 1000 r/min for 5 minutes, remove supernatant under negative pressure. Resuspend the cells with ES-DMEM and spread them onto 10 cm Petri dishes with feeder layer. (3) Replace the ES-DMEM with fresh ES-DMEM the next day. 3. ES Cell Transfection (1) Aspirate the medium under negative pressure, add 5 mL of DPBS, aspirate the DPBS and add 1 mL of Trypsin/EDTA. (2) Place the cells in the cell culture incubator at 37 ℃ for 5 minutes. Blow gently until single cell state. (3) Add 4 mL of ES-DMEM to arrest the effect of Trypsin. (4) Transfer cells to a 50 mL tube and centrifuge at 1000 r/min for 5 minutes. Aspirate the supernatant under negative pressure. (5) Resuspend the cells with ES-DMEM and spread them onto three 10 cm dishes with trophoblast cells. 1. Replace the ES cells to be transfected with fresh ES-DMEM about 3 hours before transfection. aspirate the medium under negative pressure. Add 5 mL of DPBS and aspirate off the DPBS. add 1 mL of Trypsin/EDTA. place the cells in the cell culture incubator at 37 ℃ for 5 minutes. Blow gently until single cells are present. 2. Add 4 mL of ES-DMEM to arrest the effect of Trypsin. Transfer cells to a 50 mL centrifuge tube. 3. 3. Mix 30 mL with 20 mL of Ca2/Mg*free PBS and count. Centrifuge at 1000 r/min for 5 minutes and aspirate under negative pressure to remove the supernatant (1x10cells/mL is required for electrotransformation). 4. Take 0.8 mL of Ca2/Mg2 free PBS cell suspension, add it to a 0.4 cm wide spinning cup, add 25 μg of linearized plasmid and mix well. Allow to stand at room temperature for 5 minutes. 5.240 V 500 μF electrotransfer. Allow to stand at room temperature for 5 minutes. Mix with 20 mL of ES-DMEM and spread onto 2 x 10 cm Petri dishes with feeder cells without any screening drug. 1. Selection of ES clones (1) Prepare two 96-well plates, one of which is lined with feeder cells, add 200 μl of ES screening medium, and the other add 10 μl of PBS containing calcium/magnesium ions. (2) Aspirate the medium, wash with PBS containing calcium/magnesium ions once, then add 10 mL of PBS containing calcium/magnesium ions, put it under the body microscope, gently cut a circle around the clone to be picked with a pipette, aspirate the single clone and put it into the 96-well plate containing 10 μl of PBS prepared beforehand, and repeat the procedure until 48 clones are aspirated, then aspirate the PBS, add 10 mL ES Then, remove the PBS, add 10 mL of ES medium, and place the plate in an incubator for further incubation. (3) Take another plate of clones and repeat step (2) to complete the picking of 96 clones. (4) Add 25 μl of tyipsin/EDTA to each well, digest at 37 ℃ for 5 minutes, add 80 μl of ES screening medium, terminate the digestion, gently blow into a single-cell suspension, transfer to a 96-well plate lined with a feeder, continue to incubate, and then pass on the cells when they are 80% confluent. 2. Cell Transfusion in 96-well Plate (1) Prepare three 96-well plates, two of which are lined with feeder cells and one of which is lined with only 0.1% gelatin for DNA extraction. (2) Aspirate the medium, add 25 μl of tyipsin/EDTA to each well, digest at 37 ℃ for 5 minutes, add 120 μl of ES screening medium, terminate the digestion, gently blow into a single-cell suspension, and then divide it into the three pre-prepared 96-well plates. (3) Change the solution on the next day. 3. Freeze storage of cells in 96-well plates (1) Aspirate the medium, wash with DPBS once, add 25 μl of tyipsin/EDTA to each well, digest at 37 ℃ for 5 minutes, add 80 μl of ES medium, terminate the digestion, and gently blow into a single-cell suspension. (2) Add 100 μl of pre-cooled cell freezing solution (60% ES-DMEM, 20% FBS, 20% DMSO) to each well and mix gently. (3) Add 50 μl of pre-cooled sterilized mineral oil to each well, seal the surrounding area well with sealing film, and label in detail. (4) Put the labeled 96-well plate into a pre-cooled foam box and store at -80 ℃. 4.96-well plate ES cell DNA extraction (1) ES cells in 0.1% gelatin coated 96-well plate can be used for DNA extraction after 4-5 days. (2) Aspirate the medium under negative pressure and wash twice with DPBS (200 μl per well), taking care not to wash the cells up. Add 50ul of lysis buffer to each well (add 1 mg/mL Proteinase K to the lysis buffer before use), and leave it in the wet box at 55 ℃ for 48 hours. (3) Carefully add 100 μl NaCl-ethanol (150 μl 5 mol/L NaCl to 10 mL of pre-cooled 100% ethanol) to each well. Allow to stand at room temperature for 5 hours. (4) Carefully invert the 96-well plate onto blotting paper (if you see floating DNA, only aspirate one well at a time with a gun. (Same as below). (5) Carefully add 150 μl of 70% ethanol to each well, invert onto blotting paper and wash 3 times. Invert the 96-well plate and dry at room temperature for 1~2 hours. (6) Add 1xTE (30 μl per well), seal the 96-well plate with a sealing film to prevent evaporation of the liquid, and let it stand for 24 hours, then it can be used for PCR detection (if it is not detected immediately, it can be stored at -20 ℃). 5. Positive clone amplification (1) Remove the 96-well plate where the correct homologous recombinant clone is located from -80 ℃. Rapidly rewarm the plate in a 37 °C incubator until the ice is almost completely melted. (2) Wipe the outer surface with 70% ethanol. (3) Transfer the correct homologous recombinant clone to a 24-well plate with a feeder layer of cells and change the solution the next day. (4) When the ES cells have grown to 80%-90%, transfer to two 6-well plates with feeder cells. Be careful to change the solution. (5) When ES cells reach 80% to 90%, transfer to two 10 cm cell culture dishes lined with feeder cells. Be careful to change the fluid. (6) When the ES cells reach 80% to 90%, one of the 10 cm cell culture dishes is used for freezing, and the other is continued to be cultured for 2 to 3 days for genomic DNA extraction and southern blot detection to determine the correct homologous recombination. (7) The correct ES cell clones were expanded for subsequent blastocyst injection and then transplanted into the uterus of a pseudopregnant female mouse to obtain chimeric mice. 1. Supernumerary ovulation 2. Mating and checking for plugs (1) Fertilized egg donor mice: Immediately after HCG injection, female mice will be placed in cages to mate with male mice, and the negative bolus will be detected in the morning of the next day. A record will be made of the embryos at the 8-cell stage on the morning of the 6th day (0.5 days after the egg is seen). (2) Pseudo-pregnant females: On the afternoon of day 5, females over 6 weeks of age (>20 g) were mated with ligated males in a joint cage, and the method of checking for embolism on the morning of day 6 was the same as that for donor mice. Record the bolus on the day of bolting (the day of bolting is 0.5 days, and we transferred blastocysts on the afternoon of the 8th day, i.e., on the day of bolting 2.5 days). 3. Obtaining 8-cell stage embryos On the morning of the 6th day, the donor mice were killed by neck dissection on the 2nd day of bolus. The backs of the donor mice were wetted with 70% ethanol, and the skin was lifted up with forceps at the tail end of the back, and the scissors were used to make a transverse incision. The skin at the incision was peeled away from the head side with forceps, and the muscles of the dorsal lumbar region were exposed, and the small scissors were used to make an incision, and the fat on the ovaries in the body was held in the forceps, which was dragged out of the body, and the uterus was held in the uterus, and the fat on the uterus was taken away from the uterus with the forceps. Forceps were used to hold the uterus, and the fallopian tubes and upper and middle parts of the uterus were taken and placed into a pre-prepared drop of M2 (or D-PBS). A needle is inserted into the umbilicus of the fallopian tube and the entire fallopian tube and upper and middle parts of the uterus are flushed. Collect the embryos and put the eggs into the pre-prepared M2 (or D-PBS) droplet to wash clean, wash away the blood cells and residues, etc. Transfer the washed embryos into the M16 droplet covered with mineral oil, and then put them into 5% CO2, 37 ℃ incubator for two days to the blastocyst stage. 4. Blastocyst injection (1) Prepare 100~200 μl ES-medium droplets by dividing 3~4 drops into 3.5 cm Petri dishes and cover with mineral oil, and place them in 5% CO2, 37 ℃ incubator. (2) Prepare 100 μl of injection medium (ES-medium + Hepes + 2 μl of DNase I), make a drop of about 2 mm in a 3.5 cm Petri dish, add about 15 embryos and ES cells to the droplet with a pipette and cover with mineral oil, and then pre-cool the droplet in a refrigerator at 4 ℃ (to prevent wall attachment). Pre-cool in 4℃ refrigerator (to prevent wall attachment). (3) Load the egg holding needle and injection needle into the microinjector and place the injection needle into the manipulation droplet by manipulating the microinjector. (4) Use the needle to select 10-15 small, bright ES cells with smooth edges. (5) Fix the embryo with an egg-holding needle so that the inner cell mass is fixed at the 6 or 12 o'clock position, and move the needle to the center of the field of view, adjusting the injection needle so that it is on the same plane. (6) Slightly rotate the embryo with the needle to find the cell gap for needle insertion, and quickly poke the needle several times until the needle is inserted. In this way, it is easy to insert the needle and less damage to the embryo. (7) After the needle enters the blastocyst cavity, gently blow the ES cells into the blastocyst cavity. (8) After the injection, the needle should be withdrawn slightly to prevent the injected ES cells from being pressed out by the high intra-embryonic pressure. (9) Transfer the blastocyst injected with ES cells into the ES-medium for 4 to 6 hours for recovery (the blastocyst cavity will bulge again). In addition to blastocyst injection, in recent years, the technique of 8-cell stage injection has come to the forefront. 8-cell stage injection involves the introduction of modified ES cells into the zona pellucida by means of microinjection, followed by the transfer of this chimeric embryo. Basically, a laser or other technique is used to open a hole in the zona pellucida of an 8-cell stage embryo, and an injection needle is used to introduce ES cells through this hole. The advantage of this technique is that it produces F0 mice with almost 100% germline inheritance, probably due to the rapid seizure of developmental initiative by ES cells at a more mature stage of development. Although this technique is complicated to perform, it has a promising application. Uterine Embryo Transfer About 15 embryos are transferred to the uterus of the recipient on day 6 (both sides are transferred to increase pregnancy). (1) Uterine transplantation procedure: Place the mice in an open ultra-clean table, anesthetize them with 1% sodium pentobarbital intraperitoneally, trim the hair, and disinfect the skin with 70% ethanol; make a longitudinal incision of the skin and the abdominal wall near the midline of the back at the level of the last ribs, make a small incision to cut open the peritoneum above the white fat body, and then use blunt forceps to hold the fat body and pull it out from the incision, and then pull out the ovaries, fallopian tubes, and uterus, and then carefully transfer it into the body under the microscope. transferred under the stereomicroscope. (2) Preparation of embryos for transfer: Use a hard glass capillary to make a transfer tube with a diameter of 150-200 μm. First, add liquid paraffin to the transfer tube up to the shoulder, then inhale a small bubble, then inhale the culture fluid, followed by a bubble. At the end of the tube, which is about 0.5 cm long, 15 embryos and a small amount of culture medium are inhaled. The transfer tube containing the embryos is fixed to a fixture on the stereomicroscope to avoid vibration and touching it. (3) Uterine transfer of embryos: the upper end of the uterus is gently held with blunt forceps and a small hole is made in the uterine wall beneath it with a needle. Take care to avoid blood vessels and ensure that the needle is inserted into the uterine cavity and not into the uterine wall. The needle is gently withdrawn and the transfer tube is inserted about 0.5 cm along this hole and the tube is gently blown to move the embryo into the uterus. The uterine ovary tubes are pushed back into the abdominal cavity, reset, sutured, and the skin sterilized once with 70% ethanol. (4) Resuscitation: Put the mice after transplantation back into the cage and keep the cage warm on the hot table. Set the temperature of the hot table to 37 ℃. Until the mice wake up, return the mice to the animal room. After backcrossing generation by generation, we can obtain heterozygous mice with pure genetic background, and then obtain pure heterozygous mice by crossing between heterozygotes. According to Mendelian inheritance, 25% of the offspring will be pure. This can be identified and screened by PCR. For more product details, please visit Aladdin Scientific website.
① Petri dish, 96-well plate
② Microscope
③Centrifuge
④37 ℃, 5% CO
2
④37℃, 5% CO2 incubator
⑤FACS9 flow cytometric analyzer
⑥Electrocytometer
⑦ Microinjector
Reagents:
①293 FT cells
② MilliQ water at pH 7.4, 10% glycerol, PBS with calcium/magnesium ions
③ES-DMEM
④M2 culture medium, M16 culture medium, ampicillin, kanamycin LB agar plate
⑤ Trypsin/EDTA
⑤ Trypsin/EDTA ⑥ Trypsin-EDTA solution
⑦Hanks equilibrium solution
⑧10% FBS-DMEM
⑨ Freezing solution: 95% FBS + 5% DMSO, liquid nitrogen