Stochastic Mutagenesis based on Chemical ENUs

Summary

ENU is an alkylating agent that delivers ethyl to oxygen or nitrogen groups on DNA, resulting in base mismatches or substitutions (primarily inducing point mutations, i.e., base pair substitutions at A-T. ENU has been successfully used to obtain a variety of alleles of a single gene. These include allele function, deletion of function of subequivalent alleles, and acquisition of functional mutations. Allele series at a single locus are very powerful in further determining gene function. In addition, phenotype-driven ENU screening provides tools to resolve developmental and biochemical pathways.

Operation method

Stochastic mutagenesis based on ENU of chemical substances

Materials and Instruments

Equipment:
① Disposable sterile gloves; 35-, 60-, 100-, and 150-mm cell culture dishes; disposable pipettes; 15 mL Falcon centrifuge tubes; and cryopreservation tubes;
②Surgical forceps
③Syringes, pipette guns, gun tips, etc.
④PCR instrument, water bath, centrifuge, etc.
⑤Electrophoresis instrument, electrotransfer apparatus
Reagents:
①Materials: mice
② Gonadotropin
③DNA polymerase, restriction endonuclease, ligase, trypsin, etc.
③ DNA polymerase, restriction endonuclease, ligase, trypsin, etc. ④ Agarose, electrophoresis buffer, resuspension solution
⑤ X-gal
⑤ X-gal ⑥ PBS, TRizol, chloroform, isopropanol
⑦RACE kit
⑧ DMEM high glucose medium + L-glutamine + sodium pyruvate; β-mercaptoethanol; FBS for ES cells; α-basic medium; LIF; gelatin

Move

The basic process of gene capture technology can be divided into the following steps:

I. Screening of ENU chemical mutagenesis and mutant mice based on the overall mouse level(i) Preparation of ENU

Weigh ENU in a fume hood. Weigh 100 mg of ENU in a 15 mL Falcon centrifuge tube and add 10 mL of ice-cold Soerensen buffer to prepare 10 mL of 10 mg/ml ENU solution (note that the stability of ENU is pH-dependent). Dissolve ENU by vigorous shaking. The solution is normally bright yellow, otherwise the reagent will not be usable. The concentration of ENU can be determined using a 398-entry spectrophotometer, using the formula ［ENU］＝(OD1/0.72)］ to calculate the concentration of ENU. The formula for calculating the volume of ENU for injection is VENU (mL) = (Dose/[ENU] Body Weight (g)).The ENU dose range is 200 to 400 mg, with a maximum of 200 to 250 mg for a single treatment, and 100 mg for multiple treatments, up to a maximum of four treatments (i.e., 400 mg). If 06-BG treatment is to be performed, dissolve 06-BG with DMSO and dilute it to 10 μmol/L with ES cell culture medium together with the appropriate concentration of ENU, which should be used immediately after filter sterilization.

(ii) Mutant Mouse Preparation

1. Breeding and strain of mice

(1) SPF environment: The optimal experimental environment for mutation in mice is a specific pathogen-free (SPF) environment. Since ENU causes deletion of several stem cell types, such as hematopoietic stem cells, this can also be produced by temporary pathogen sensitization. As a result, injected male mice not only undergo a period of sterility, but they also die earlier due to having a higher bacterial susceptibility than untreated mice.

(2) Selection of mouse strains: Selection of appropriate mouse strains is an important prerequisite for effective mutation screening.

2. Preparation of mutant mice by ENU injection

(1) Preparation before injection

(1) During injection, gloves, overalls, goggles and mask are required.

(2) Prepare some alkaline solution (0.1 mol/L NaOH) on the working table. Once ENU spills out, treat it with alkaline solution immediately and clean it up after a few minutes of alkaline reaction.

(2) Injection procedure

(1) Mice were injected intraperitoneally with 0.5 mL of ice-cold ENU solution (concentration according to the average body weight of each group). The injection procedure is performed as described on Mouse Embryo Operations.

2) The injection should be completed within 60 minutes of ENU preparation.

3) If there is any ENU remaining, it must be disposed of immediately with 0.1 mol/L NaOH.

4) After ENU injection, the chamber must be closed for at least 24 hours to minimize the risk of exposure to mutagens.

(3) Infertility assay: For the infertility assay, each mutant-treated male mouse was paired with a wild-type female mouse 7 weeks after the last injection to ensure that the offspring analyzed for these mutations were derived from cells exposed to the spermatogonial stem cell stage. In contrast, mouse spermatogenesis from spermatogonial stem cells to mature spermatozoa takes approximately 49 to 51 days. The infertility cycle is dependent on the success of the proliferation of new spermatogonia in the seminiferous tubules. Since there is some correlation between the sterility cycle and the mutation frequency, the phenotypic analysis of the complete F1 generation will not be performed until 80 days after injection.

(4) Breeding Mice for Dominant Mutation Screening

(1) The breeding process for dominant mutation phenotype screening in mice is shown in Figure 5-4-3. The basic steps are as follows: (1) After a sterile period of about 80 days, fertile mutation-treated male mice are paired with wild females to produce the offspring, G1, and it is assumed that they are both heterozygous for the mutation. (ii) Each male mouse was paired with a group of two (or more) females, and one week later the males were paired with another group of females, and so on to accelerate the number of G1 generations. (iii) The number of G1 offspring from each mutation-treated mouse must be limited to 100, because only a small number of spermatogonial stem cells may survive in the mouse to produce clusters of mutations.

(2) Heritability of dominant traits: ① Mutant candidate mice in G, are paired with wild-type mice to produce G2 generations to test the heritability of the mutant phenotype. (ii) After collecting 20 G2, the phenotype of interest is tested. This number is sufficient to ensure that a fully epistatic dominant mutation is P = 0.01 valid. (iii) The mutation can be confirmed if one of the offspring exhibits the mutant phenotype. (iv) A dominant mutant line is created through a distant cross between the heterozygous (+/-) and original selfing lines. Since only 50% of the progeny carry the mutation, phenotypic evaluation is performed on the entire progeny. ⑤ If two heterozygotes with the phenotype are crossed to produce a pure (-/- or M/M) mouse it may be a subdominant mutation. If there is a gradual phenotypic difference between a heterozygote and a pure heterozygote, then it can be concluded that this is a subdominant trait.

(5) Breeding of recessive mutation screening mice: The process of breeding recessive mutant mice is shown in Figure 5-4-3. Since it is much faster to produce G3 with G, male than with G, female, we will only discuss this faster strategy. And there are data suggesting that obtaining recessive mutant mice is effective at P = 0.005. When selecting G, candidate mutant mice to establish recessive mutant lines, one needs to consider that (1) G, males are all considered heterozygous for the mutation but do not have a distinct phenotype, and (2) each G, builder should come from a different mutation-treated male to establish a broadly variant recessive phenotype.

(1) Breeding strategy for recessively inherited male G1 mice: (i) male G1 group builders (+/-) are crossed with a group (4) of wild-type females to produce G2 offspring. Only female G2 (+/- or +/+) were collected here. (2) Female G2 (8) were backcrossed with G1 to produce 40 G3 offspring, who will be used for the evaluation of recessive mutations.

(2) Breeding strategy for recessive female G1 mice: ① A female G1 builder (+/-) is crossed with a wild-type male to produce G2 offspring (+/- or +/+). Mice of both sexes are collected. (ii) At least 4 groups of G2xG2 crosses are established to produce G, offspring, noting that only 8.3% (1/12) of the G's here, may possess the recessive mutant phenotype.

3) Heritability testing for recessive genetic phenotypes. The heritability of the mutant phenotype was tested using the following two-step method: ① G, pure (-/-) candidate mice were crossed with wild-type (+/+) mice to produce heterozygous G4 (+/-) offspring. (ii) Establishment of 2 sets of G4 G4 crosses to produce G3 progeny, with at least 20 G3s used for phenotyping. This data is valid for a full-exploitation mutation that produces 25% of the predicted phenotype. ③ If one offspring here exhibits the mutant phenotype, the mutation can be confirmed. ④ A new recessive mutation is established by crossing two phenotyped mice. To test the fertility of the recessive mutation, a phenotyped (-/-) male and female mouse were crossed with the original wild-type mouse, respectively. If they are purebreds, they may happen to be sterile in one sex. At this point it is necessary to switch from a pure (-/-) to a heterozygous (+/-) breeding strategy.

(6) Genetic mapping: the localization of chromosomes that have been mapped is used to clone a gene or to test for certain pre-existing genes. If the mutant allele is fully dominant or if the mutant allele is recessive but the pureblood is fertile, it is better to use the backcross strategy. If pure individuals are sterile and have a recessive epistatic mutation, an intercross strategy is preferred. The best conditions for linkage analysis are to cross two different mouse lines with high polymorphisms. Intercross analyses were performed using a set of genome-wide microsatellite markers for genetic mapping (www.informatics.jax.org) or, more recently, single-nucleotide polymorphism (SNP) for genetic mapping ( www.genome.wi.mit.edu/SNP/mouse/ ). mouse/).

(7) Construction of mutation database

II. ES cell-based ENU mouse mutation technology(I) Mutation of ES cells into ENU

1. ES cells were spread on a 100 mm cell culture dish with feeder layer of cells and cultured at 37 ℃ under 5% _CO2. 2.

2. Cultivate the cells until they reach 80% polymerization.

3. Replace the medium with 10 μmol/L 06-BG ES cell culture medium and incubate at 37 ℃ under 5% _CO2 for 12 to 16 hours.

4. Remove the ES cell culture medium and wash the cells with 1xPBS, add 3 mL of 0.05% trypsin, and incubate for 10 minutes at 37 ℃ with 5% _CO2.

5. Gently blow the cells with a pipette to form a single-cell resuspension.

6. Add 10 mL of ES cell culture medium and centrifuge at 700 g for 5 min.

7. Remove supernatant and resuspend precipitate with ES cell culture medium to bring cell concentration to 5x10/mL, add 10 μmol/L 06-BG and specific concentration of ENU (0.5 mg/mL).

8. Incubate the resuspended cells at 37 ℃ with uniform shaking for 1 hour (2 hours). 9.

9. Wash the cells and count the cells. Cells are finally divided into 3 parts: cell death assay, cell mutation rate assay, expression and freezing.

(ii) ES cell death assay

1. Inoculate 1x10 mutagent-treated cells and untreated control cells in 60 mm dishes with feeder layer cells. 2.

2. Cultivate the cells as usual and count the total number of cell clones after 1 week.

3. Compare the treated and control groups and estimate the cell survival rate.

(iii) Detection of cell mutation rate

1. Inoculate half of the mutagen-treated cells in 60 mm dishes (without feeder layer). 2.

2. Normal passaging culture the cells for about 10 days.

3. 1.5 mL of 0.05% trypsin digest the cells and incubate them for 10 minutes in a cell culture incubator.

4. Gently blow the cells into single cell resuspension. 5.

5. Wash cells with ES cell culture medium and inoculate 0.5x10° cells into 4 150 mm dishes. Add 6TG to a final concentration of 100 μg/mL. 3 days later, remove the selection medium and count the number of clones two weeks later.

6. Simultaneously inoculate 1x10 cells in 60 mm dishes without 6TG treatment and count the number of clones after 1 week of incubation; these cells will be used to determine cell survival in culture.

7. Calculate the mutation rate.

(iv) Preparation of mutant chimeric mice(v) Screening for mutations(vi) Localization of mutant genes Unlike gene targeting or insertion mutagenesis, mutant mice induced by ENU have no molecular markers. Identification of mutant genes can be accomplished by localization of clones or candidate genes.

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