5. Determine the live weight of eac

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:
Formula

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

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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:Formulawhere 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) × XThe 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.Animals may appear wobbly and lethargic for ~30 min after injections because of the alcohol. It should take ~1 h to inject 30 mice.Inactivation and Disposal of ENU8. 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 Mutations12. 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.The breeding scheme will depend on the type of mutation being studied, i.e., dominant, recessive, or modifying (see Discussion). Plan to recover about 50 gametes per male for dominant or modifying mutations, and about 30 gametes per male for a recessive scheme.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.

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