Generating Gene Knockout Mice for Studying Mechanisms Underlying Drug Addiction - Drugs of Abuse: Neurological Reviews and Protocols - page 350

perform PCR for such verifi cation. Once verifi ed, ES cells are ready for injection
( see Notes 7 and 8 ).

3.3. Knockout Mice
The goal for this step is to introduce the ES cells harboring proper homolo-
gous recombination into mouse blastocysts to generate male chimeric mice. If
the injected ES cells, which are of 129 origin, contribute to the development
of a mouse, the coat color of the mouse may be chimeric which can be easily
visualized. Each injection process takes 5 d. Then, the chimeras are bred
repeatedly with C57BL/6J mice to obtain mice homozygous for the desired
gene mutation.

3.3.1. Establishing a Mouse Colony
Two groups of mice are required. One group is for blastocyst production. For
traditional reasons, C57BL/6J inbred strain of mouse is used. Forty male stud
mice 6-16 wk of age and 160 female mice 4-8 wk of age are needed. Another
group is to provide foster mothers. Twenty vasectomized C57BL/6J × DBA2 F1

males 6-16 wk of age and 80 normal C57BL/6J DBA2 F1 females 10-18 wk

of age are needed. This steady-state colony size is suffi cient to support three
injections per week.
3.3.2. Blastocysts and ES Cell Preparation

×

1. Timed breeding: On d 1, select 20 C57BL/6J female mice that are in estrous
cycle and breed with C57BL/6J male mice individually between 4:00 and
6:00 P . M . On d 2, check the female C57BL/6J mice for vaginal plugs before
11:00 A . M . On the same day, select 10 C57BL/6J DBA2 F1 female mice that

are in estrous cycle and breed with individual vasectomized C57BL/6J

×

DBA2
F1 male mice between 4:00 and 6:00 P . M . On d 3, check plugs for the C57BL/
6J

×

DBA2 F1 females before 11:00 A . M . More than 50% of the females are
plugged on the average. The plugged C57BL/6J females and C57BL/6J

×

DBA2
F1 females can be group-housed separately. If a higher yield of blastocysts is
desired, superovulation can be performed.

×

2. ES cell preparation: Treat three wells in a 12-well tissue culture dish with 0.1%
gelatin for 20 min at room temperature. Thaw one vial of amplifi ed ES cells from
Subheading 3.2.5. , resuspend in 9 mL of ES cell medium, centrifuge at 2000 g ,
resuspend in 3 mL of ES cell medium, plate out in three wells in the 12-well
dish, and incubate at 37°C in 5% CO 2 . No EF feeder cells are needed. On d 4,
change medium for the ES cells. On d 5, change medium again and ES cells
should be about 30-60% confl uent. Just before the injection, remove culture
medium, trypsinize for 30 s and resuspend in 1 mL of ES cell medium one
well at a time.

3. Blastocyst isolation: On d 5, set up a 25-gauge needle with a 10-mL syringe and
a 50-mm Petri dish with two drops of ES medium covered with dimethylpolysi-