Authors: Andrew P. Salinger and Monica J. Justice1
Corresponding author ([email protected])
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 × 10e−3 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.
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.
3.Transfer 400 μL of the suspended ENU to a disposable plastic cuvette and bring the volume to 2000 μL with phosphate/citrate buffer (a 1:5 dilution). Prepare a 1:50 dilution of 95% ethanol in phosphate/citrate buffer (also in a plastic disposable cuvette) to use as a blank. Determine the OD398nm of the ENU relative to the blank.
4.Calculate the concentration of the ENU in solution based upon the observation that a 1 mg/mL solution gives an OD398nm of 0.72.
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).
5.Determine the live weight of each mouse prior to each injection and record. Mark each animal to distinguish it on subsequent days.
6.Calculate the volume of ENU for injection depending on the desired dose. For 100 mg ENU/kg mouse body weight, the calculation is as follows:
where 0.72 is OD398nm at 1 mg/mL and 5 is the dilution factor (see Steps 3-4).
Volume of ENU to inject (mL) = mouse weight (g) × X
The concentration of the ENU solution should be such that <1 mL is injected into each animal.
Various inbred strains of mice react differently to a given dose of ENU. Likewise, different doses of ENU give different mutation rates. To produce the highest mutation rate, an appropriate amount for C57BL/6J mice is three weekly doses of 100 mg/kg body weight. An effective single dose is 250 mg/kg. Protocols for optimizing doses for different inbred strains are referenced in the Discussion.
7.In an efficient chemical fume hood, administer the appropriate amount of ENU intraperitoneally to each mouse.
Inactivation and Disposal of ENU
8.Clean all spills and soak all equipment (beakers, vials, etc.) and gloves coming into contact with ENU with inactivating solution. Pull up inactivating solution into all needles that have been in contact with ENU. Put treated items in a plastic bag and discard in waste. Inactivate any remaining ENU solution and discard appropriately.
9.Inject at least 50 mL of inactivating solution into the remaining ENU in the ISOPAC container. Leave in a chemical fume hood, exposed to light, for at least 24 h.
10.Remove the seal from the ISOPAC container and discard the inactivated ENU into a chemical hazardous waste container. Record appropriately. Rinse the ISOPAC container with H2O, and discard the contents into a chemical hazardous waste container. Discard the emptied ISOPAC container in glass waste.
11.Keep mice in an efficient chemical fume hood for at least 24 h after injections, or change bedding at least 24 h after injections into a plastic bag containing paper saturated with inactivating solution. (This usually requires marking the pens containing treated animals.)
Mating Males to Recover Mutations
12.After injection, males go through a period of infertility that lasts ~10-12 wk. Set up males in cages with females 8-10 wk after the last injection to assess recovery of fertility. See Troubleshooting.
13.Select an appropriate breeding scheme, according to the mutation of interest.
14.Use rotation matings (see Fig. 1) to mate the males according to the breeding scheme chosen in Step 13. Mate the males to a new female (or two new females, depending upon animal protocol) each week for 6-7 wk, until the appropriate number of gametes has been obtained. Set up each new cage as it is needed each week.
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.
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’s 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).