Mutagenesis of genomes using chemical mutagens

Summary

Currently, there are two main strategies for studying gene function using model mice: the gene-driven approach and the phenotype-driven approach. Gene knockout is a typical gene-driven approach, which takes about 1.5 years to produce a knockout mouse and can only manipulate genes with known sequences, which is not suitable for the needs of modern biology in terms of high efficiency and high throughput. Phenotype-driven method refers to the use of chemical reagents to induce a large number of random mutant phenotypes in animals in a relatively short period of time, or through transposon technology to generate mutations in the genome as a whole, so as to obtain animals with special phenotypes through screening and to identify their genetic basis.

Principle

The basic principle of genome mutagenesis is that spontaneous mutations are generated by base mismatches, deletions or duplications in the processes of DNA replication, gene transcription and DNA damage repair; induced mutations are usually caused by a variety of mutagens.

Operation method

Mutagenesis of model organism genomes using ENUs

Materials and Instruments

Reagents:
Acetyl nitrosourea, mice,
Apparatus:
Incubator, fume hood,

Move

The basic process of using ENU to mutagenize the genome of a model organism can be divided into the following steps:

1. Preparation and dilution of ENU solution ENU is very sensitive to light, humidity and pH. Before each injection, a new package of ENU should be dissolved and sealed with aluminum foil to protect it from light, and the injection should be made within 3 hours after dissolution. Handling should be carried out in a fume hood and the operator should wear protective clothing, plastic gloves and a mask. Inject 10 ml of 95% ethanol directly into the ISOPAC container containing ENU using an 18 G needle and syringe. Warm the bottle in the palm of your hand and shake gently until the ENU is completely dissolved (about 10 minutes), the solution should be a clear yellow color. Inject another 90 ml of phosphate-citrate buffer into the vial and mix thoroughly. If a less concentrated solution is to be prepared, 50 ml of the ENU solution can be removed from the ENU vial and added directly to 50 ml of the inactivation solution, which can be discarded after 2 hours. Inject 50 ml of phosphate-citrate buffer into the remaining ENU solution to obtain a 1-fold dilution of ENU solution.

2. Determination of the concentration of the ENU solution is not accurate in purchased ENU containers and must be determined empirically for each portion of the prepared ENU solution. A 400:1 solution of ENU was added to a disposable plastic cup, and the volume was expanded to 2000:1 with phosphate-alkanoic acid buffer, while 95% ethanol was diluted 1:50 with phosphate-alkanoic acid buffer in the disposable plastic cup (blank control). The OD value of the ENU solution at 398 nm is measured spectrophotometrically. 0D398 for l mg/ml of ENU solution is 0.72. To ensure accuracy, the ENU solution can also be scanned in the wavelength range 350 - 450 nm. The peak should be at 398 nm. 3.

3. Inject mice with ENU either as a single injection or in separate injections, depending on the purpose of the experiment and the strain of mouse. Injections should be performed in a fume hood. Male mice should be fully mature (8 weeks of age or older) prior to injection. Split injections should be given on the same day of the week for approximately 3 weeks, preferably starting when the mice are 8-9 weeks old. Mice are weighed prior to injection and the dose is determined based on the body weight of the mouse, with 100 mg per kilogram of body weight being injected in a volume of <l ml. Different strains of mice respond differently to ENU, resulting in different mutation rates. To maximize the mutation rate, C57BU6 mice can be injected with 100 mg/kg body weight, intraperitoneally once a week for 3 injections, or a single injection of 250 mg/kg body weight. Due to the effects of ethanol, the animals will be somewhat shaky for 30 minutes after injection. It takes approximately 1 hour to inject 30 mice.

4. Inactivation of ENU Residual ENU solution must be inactivated before discharge ENU has a short half-life in alkaline environments and can be inactivated with 0.1 mol/L alkaline sodium thiosulfate. All spills of ENU solution during the experiment should be wiped up with the inactivation solution; all equipment in contact with ENU should be immersed in the inactivation solution; syringes and pipettes should be filled with the inactivation solution and packaged in a plastic bag to be discarded after disposal. Inject at least 50 ml of inactivation solution into the remaining ENU solution in the ISOPAC container and place in a fume hood and expose to light for at least 24 hours. The inactivated ENU solution is collected in a "hazardous chemicals" container and registered. Wash the remaining ISOPAC container with water, pour the liquid into the "Hazardous Chemicals" container and discard the ISOPAC container. Mice should be placed in an open fume hood for at least 24 hours after injection and bedding changed 24 hours after injection into a plastic bag with absorbent paper saturated with the inactivation solution. 5.

5. Mice are mated to recover the mutation Injected male mice are sterile for 10 - 12 weeks. At 8 - 10 weeks post-injection, injected male mice are caged with female mice to observe recovery of fertility. Mating is designed according to the needs of the study (for dominant and modified mutations, the design is to recover approximately 50 gametes per mouse; for recessive mutations, the design is to recover 30 gametes). Mating is done in male rotation: male mice are mated with 1 - 2 new females per week for 6 - 7 weeks until a sufficient number of gametes are obtained.

Caveat

PrecautionsENU is a toxic reagent and should be handled in strict accordance with the requirements for protection against chemical poisons.

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