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Protocol:Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU)
【字体: 大 中 小 】 时间:2008年04月03日 来源:生物通
编辑推荐:
一种用ENU诱变来检测小鼠基因组的方法。
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
1Corresponding author (mjustice@bcm.tmc.edu )
INTRODUCTION
This protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 x 10? in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.
MATERIALS
Reagents
Ethanol (95%)
Inactivating solution
ENU has a very short half-life under alkaline conditions. Prepare one of the following solutions for ENU inactivation:
0.1 M KOH (5.6 g KOH pellets dissolved in 1000 mL H2O)
Mice (males of appropriate strain; 8-12 wk old), for injection
N-ethyl-N-nitrosourea (ENU) (1-g ISOPAC containers; Sigma N3385)
The labeling of 1 g of ENU per container is only approximate. The actual amount per container ranges from about 0.7 g to 1.2 g (see Steps 3-4).
Equipment
Bags (plastic)
Chemical fume hood
Cuvettes (plastic disposable)
Foil
Glass waste container
Hazardous waste container (chemical)
Needle (18-gauge)
Paper for changing bedding (optional; see Step 11)
Protective clothing for handling ENU (plastic gloves, lab coats, and masks)
Scale for weighing mice
Spectrophotometer
Syringes
METHOD
Preparation of ENU Solution
ENU is very sensitive to light, humidity, and pH. Dilute a new container of ENU prior to each weekly injection and protect from light using a foil wrap. Inject mice (Step 7) within 3 h of diluting the ENU.
Dissolving and Diluting ENU
Determining Concentration by Spectrophotometry
Because the amount of ENU per container varies, the concentration of each prepared ENU solution must be determined empirically. Measurement of concentration also controls for dilution errors.
Injection of Male Mice with ENU
Injections may be a single dose of ENU or a fractionated dose. Determine which is appropriate for your experiment and strain of mice. Fractionated doses are administered weekly at approximately the same time each week.
Males should be fully sexually mature (8 wk) prior to injections. If a fractionated dose is used, the experiment will take 3 wk; thus, it is important to begin the experiment with 8- to 9-wk-old males (not older mice).
Inactivation and Disposal of ENU
Mating Males to Recover Mutations
View larger version (27K): [in a new window] |
Figure 1. A schematic diagram showing rotation matings. Obtaining the appropriate number of gametes from each treated male is important for mutagenesis. To obtain enough gametes before ENU-treated males succumb to illness (in particular, the commonly used C57BL/6J strain is susceptible to the development of T-cell lymphoma), rotation matings are advised. Place each male into a new cage with one or two females each week for 6- wk, depending upon how many gametes are needed from each male. If two females are used, a pregnant female can be placed into a separate cage for bearing her litter, if required by the institutional animal protocol. The rotation breeding strategy also improves efficiency of weaning and mutation recovery. Females are shown in white; males are shown in black. |
TROUBLESHOOTING
Problem: The ENU does not dissolve.
[Step 1]
Solution: Use 95% ethanol as the solvent, and be sure that the ENU is completely dissolved in ethanol before adding the phosphate/citrate buffer.
Problem: Males do not recover fertility.
[Step 12]
Solution: Consider the following:
Problem: Males do not produce mutant offspring.
[Step 14]
Solution: ENU is a powerful mutagen and mutations should be recovered. Dominant mutations should occur at a rate of one visible dominant mutation in every 100 offspring in the first generation of matings. Consider the following:
DISCUSSION
ENU treatment of male mice is a simple and effective method for obtaining mutations in a forward genetic screen (Guenet 2005; Russell et al. 1979), and is now established as part of a mouse geneticist抯 toolkit. The method described here was designed to produce the highest mutation rates while avoiding toxicity (Russell et al. 1982). However, some knowledge of genetics is required in order for the protocol to be carried out effectively, so consult papers and textbooks for breeding schemes (Justice 1999). For example, many successful screens for dominant mutations have been conducted (Hrabe de Angelis and Balling 1998). Likewise, screens for recessive mutations using three generation pedigree breeding schemes or balancer chromosomes have been successfully carried out (Kasarskis et al. 1998; Herron et al. 2002; Kile et al. 2003). Banks of sperm and DNA samples from mutagenized males are useful for identifying point mutations in specific genes (Coghill et al. 2002) and, because of the advances in sequencing technology and mutation detection, molecular identification of point mutations is straightforward. Screens for modifying mutations are simple and effective in a small laboratory setting with limited amounts of mouse space, and are likely to be the most common use of forward genetics in the future (Carpinelli et al. 2004). Choosing the appropriate dose of ENU, according to experiment or strain background (Step 6), is very important (Justice et al. 2000). Although many strains, including C57BL/6J, can handle a high dose of ENU, an appropriate dose for FVB/N mice is only a single injection of 150 mg/kg body weight (Russell et al. 1979; Davis et al. 1999).
REFERENCES
Carpinelli, M.R., Hilton, D.J., Metcalf, D., Antonchuk, J.L., Hyl, C.D., Mifsud, S.L., Di Rago, L., Hilton, A.A., Willson, T.A., Roberts, A.W., et al. 2004. Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling. Proc. Natl. Acad. Sci. 101: 6553?558.
Coghill, E.L., Hugill, A., Parkinson, N., Davison, C., Glenister, P., Clements, S., Hunter, J., Cox, R.D., and Brown, S.D. 2002. A gene-driven approach to the identification of ENU mutants in the mouse. Nature Genet. 30: 255?56.[Medline]
Davis, A.P., Woychik, R.P., and Justice, M.J. 1999. Effective chemical mutagenesis in FVB/N mice requires low doses of ethylnitrosourea. Mamm. Genome 10: 308?10.[Medline]
Guenet, J.-L. 2005. Chemical mutagenesis of the mouse genome: An overview. In Genetica: Mutagenesis of the mouse genome (eds. M. Justice and M. Bedell), pp. 9?4. Kluwer Academic Publications, Dordrecht, The Netherlands.
Herron, B.J., Lu, W., Rao, C., Liu, S., Peters, H., Bronson, R.T., Justice, M.J., McDonald, D., and Beier, D.R. 2002. Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis. Nature Genet. 30: 185?89.[Medline]
Hrabe de Angelis, M. and Balling, R. 1998. Large scale ENU screens in the mouse: Genetics meets genomics. Mutation Res. 400: 25?2.[Medline]
Justice, M.J. 1999. Mutagenesis of the mouse germline. In Mouse genetics and transgenics: A practical approach (eds. I. Jackson and C. Abbott), pp. 185?15. Oxford University Press, Oxford, UK.
Justice, M.J., Carpenter, D.A., Favor, J., Neuhauser-Klaus, A., Hrabe de Angelis, M., Soewarto, D., Moser, A., Cordes, S., Miller, D., Chapman, V., et al. 2000. Effects of ENU dosage on mouse strains. Mamm. Genome 11: 484?88.[Medline]
Kasarskis, A., Manova, K., and Anderson, K.V. 1998. A phenotype-based screen for embryonic lethal mutations in the mouse. Proc. Nat. Acad. Sci. 95: 7485?490.
Kile, B.T., Hentges, K.E., Clark, A.T., Nakamura, H., Salinger, A.P., Liu, B., Box, N., Stockton, D.W., Johnson, R.L., Behringer, R.R., et al. 2003. Functional genetic analysis of mouse chromosome 11. Nature 425: 81?6.[Medline]
Russell, W.L., Kelly, E.M., Hunsicker, P.R., Bangham, J.W., Maddux, S.C., and Phipps, E.L. 1979. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc. Natl. Acad. Sci. 76: 5818?819.
Russell, W.L., Hunsicker, P.R., Raymer, G.D., Steele, M.H., Stelzner, K.F., and Thompson, H.M. 1982. Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia. Proc. Natl. Acad. Sci. 79: 3589?591.
Caution |
Caution |
Recipe |
Sodium thiosulfate (Na2S2O3)
Dissolve 200 g Na2S2O3 and 4 g NaOH in a final volume of 1000 mL H2O.
Recipe |
0.05 M sodium citrate (pH 5.0)
Adjust to pH 5.0 with phosphoric acid and filter sterilize (22-祄 pore size filter). Prepare immediately before use.
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