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. The 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. the efficiency of DNA homologous recombination is highly correlated with the length of the homology arm, and the longer the homology arm, the more efficient the homologous recombination, and the more efficient the homologous recombination, the more efficient the homology arm. The longer the length of the homologous arm, the higher the efficiency of homologous recombination and the higher the probability of obtaining ES cell clones that have undergone homologous recombination. Chromosome recombination is commonly used in phage P1 cloning, phage-derived artificial chromosomes (P1 bacteriophage, PAC) and bacterial artificial chromosomes (BAC).
Operation method
Mouse Chromosome Recombination Technology
Materials and Instruments
Equipment: Move The main operation steps of mouse chromosome recombination technology are briefly described as follows: (1) Construction of vector (1) Cultivate DH10B containing the corresponding BAC at 37 ℃ overnight, and then pick a single clone on the next day and incubate it at 20 μg/mL chloramphenicol concentration at 37 ℃ overnight. (2) Inoculate the overnight culture medium with 4 mL of fresh chloramphenicol medium and incubate at 37 ℃ until the OD≈0.5～0.7. Cool on ice for 30 min, centrifuge at 5000 r/min and 4 ℃ for 5 min, and discard the supernatant. (3) Resuspend the precipitate with 2 mL of deionized water of pH 7.0, and then centrifuge at 4 ℃ for 5 minutes at 5000 r/min. Repeat this step 4 times. 4) Resuspend the precipitate in 70 μl of deionized water and add 10 ng of pSim18 plasmid and mix gently. Transfer to a clean, pre-cooled 0.1 cm spinning beaker. 5) Electrotransfer at 1.75 kV, 25 μF, 200 Ω, and then quickly add to SOC or LB medium. incubate at 32 ℃ with shaking for 1 hour. 6) Spread the plate with double antibody containing chloramphenicol (20 μg/mL) and thiamphenicol (100 μg/mL), and incubate at 32 ℃ for 24～48 hours. The single clone was picked and incubated at 32 ℃ with shaking. 7) Linearize the pRpsl-neo plasmid by NheI or EcoRV digestion, and then use it as a template for amplification by chimeric primers (60-80 bp homologous sequence on each side of the primer) to obtain the rpsl-neo fragments with homologous sequences on both sides. (8) Inoculate the culture medium containing BAC and pSim18 plasmids with fresh medium and incubate at 32 ℃ until OD≈0.5～0.7, then incubate at 42 ℃ with shaking for 15 min, and then put on ice for 30 min to make the electroreceptor state, and then electroreceptorize the rpsl-neo fragments (under the same conditions as above). Spread on chloramphenicol and kanamycin (15~25 μg/mL) plates and incubate at 32 ℃ for 24~48 hours. (9) Pick the single clone and incubate it in chloramphenicol and kanamycin double-resistant LB medium at 32 ℃ with shaking overnight. Pick the plasmid PCR to identify the recombinant clone, and use the appropriate restriction endonuclease digestion to identify. (2) Pure preparation of recombinant BAC vectors: amplify and pick single colonies in E.coli, BAC plasmid DNA manipulation can be modeled after standard molecular biology procedures. The main difference is to use a wide-mouth tip as much as possible to avoid vortexing and freeze-thawing the BAC DNA, and it is recommended to use QIAGEN PlasmidMaxi reagent to extract the BAC, which can be found in the QIAGEN Plasmid Maxi kit. (3) Prepare linearized BAC for microinjection: (1) Take 10-30 μg of BAC DNA, digest with BssHII, NruI or NotI, 200 μl of the system, incubate at 37 ℃ for 3 hours. 2) Melt 2 g of low melting point agarose with 200 mL of 0.5xTBE. The gel was poured in a 15 cm x 15 cm gel tank. 7 wells were spotted, 1 slightly wider and 6 slightly narrower (4 cm wide). After the gel has set, spot the digested liquid in the wide trough. Electrophoretic Marker was spotted in either slot to accurately estimate the size of the DNA fragments for microinjection. Run the gel in 0.5xTBE buffer with an initial condition of 6 V/cm and final transition times of 1 and 10 seconds, respectively, at 14 ℃ for 14 hours of electrophoresis. (3) After electrophoresis, cut off one of the lanes of the gel and stain it with EB. After 30 minutes of staining, mark the position of linearized BAC bands under UV light. Reposition the cut strip and cut off the corresponding BAC band in the unstained colloid portion. 4) Equilibrate the colloid in agarase buffer containing 100 nmol/L NaCl. 5) Transfer the colloid to a microcentrifuge tube and leave at 65 °C for 10 minutes to melt the gel in a 42 °C water bath. Add 5 U agarase (5 μl) per 200 μl of gel. Incubate at 42 °C for at least 1 hour. Place on ice for 2 minutes to solidify undigested agarose. centrifuge at 20,000 g for 2 minutes to precipitate undigested agarose. If undigested agarose is evident, repeat the digestion. 6) To estimate the concentration of linearized fragments, fluorescence methods are recommended, as is agarose electrophoresis. Dilute the DNA to a concentration of 3 ng/μl with an agarase buffer solution containing 100 nmol/L NaCl and store at 4 ℃. It was reported that there was no significant difference in transgene efficiency or transgene copy number between different concentrations of BACDNA at 3 ng/μl, 2 ng/μl and 0.5 ng/μl, respectively. For microinjection, linearized BAC in agarase buffer containing 100 mmol/L NaCl can be stored for at least 5 days. 7) Run electrophoresis with 1% gel to ensure that the fragments are correctly sized and not fragmented prior to transfection of embryonic stem cells or microinjection of fertilized eggs. 8) Follow the standard procedure for ESC transfer or microinjection of fertilized eggs. . Positive ES cell clones screened by G418 were extracted for DNA for PCR and other subsequent analyses. 2. The genomic DNA of positive ES cells was used as template for PCR to amplify the 3' and 5' ends of the BAC transgene vector, and the correctly amplified clones were used for subsequent analysis. For more product details, please visit Aladdin Scientific website.
① Petri dish, 96-well plate
② Microscope
③Centrifuge
④ Incubator
⑤PCR instrument
⑤PCR instrument ⑥Electrolyzer
⑦ Gel electrophoresis instrument
Reagents:
①DH10B (recA) Escherichia coli; pRpsl-neo plasmid or other plasmids with suitable screening markers; pSim18 plasmid; corresponding BAC clones
② kanamycin; chloramphenicol; thiamphenicol
(iii) Culture medium