3 Methods
3.1 Mouse Zygote
Preparation by In Vitro
Fertilization
Carry out mouse zygote preparations following the textbook
Manipulating the Mouse Embryo: A Laboratory Manual (CSHL
press).
1. Inject 0.1 ml of diluted PMSG (7 units/ml) into each female
by intraperitoneal injection.
2. Inject 0.1 ml of diluted hCG (7 units/ml) 46–48 h after the
PMSG injection.
3. Prepare three IVF dishes: Place a 200-μl drop of HTF in each
dish and cover the medium with mineral oil.
4. Prepare one sperm preincubation dish: Place a 100-μl drop of
CARD FERTIUP Mouse Sperm Preincubation Medium and
cover the medium with mineral oil.
5. Prepare three Wash dishes: Place three 100-μl drops of KSOM
in each dish and cover the medium with mineral oil.
6. Prepare culture dishes: Place five 20-μl drops of KSOM in each
dish and cover the medium with mineral oil. The number of
culture dishes depends on the number of mouse zygotes.
50 zygotes can be cultured in one dish (10 zygotes in each
20-μl KSOM drop).
7. Collect sperm from the epididymis and transfer it into the
sperm preincubation dish. Incubate the dish for 1 h in a
37
C incubator with 5% CO 2 .
8. Collect oocyte masses from the oviducts of the female mice
13 h after hCG injection and place them in the IVF dishes.
Oocyte masses from two female mice are placed in one
dish. Incubate the dish for 30 min in a 37
C incubator with
5% CO 2 .
9. Transfer the sperm suspension into the IVF dish and incubate
the dish for 3–5 h in a 37
C incubator with 5% CO 2 .
10. Collect and wash the presumptive zygotes with KSOM
medium in the WASH dish by mouth pipette.
11. Move the zygotes into the culture dishes. Place 10 zygote in
one 20-μl KSOM drop. Incubate the dishes until
electroporation.
3.2 Genome Editing
Solution
1. Mix 0.6 μl of 1 μg/μl crRNA, 0.6 μl of 1 μg/μl tracrRNA, and
4.2 μl of Opti-MEM medium (5.4 μl total). For the
HDR-mediated knockin, add 0.6–1.2 μl of 1 μg/μl ssODN
to the mixture (see Note 4).
2. Heat the mixed reagents at 95
C for 3 min.
3. Add 0.6 μl of 1 μg/μl Cas9 into the mixed reagents (6 μl total).
4. Place the mixed reagents described above on ice until use.
Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically. . .
123
3.1 Mouse Zygote
Preparation by In Vitro
Fertilization
Carry out mouse zygote preparations following the textbook
Manipulating the Mouse Embryo: A Laboratory Manual (CSHL
press).
1. Inject 0.1 ml of diluted PMSG (7 units/ml) into each female
by intraperitoneal injection.
2. Inject 0.1 ml of diluted hCG (7 units/ml) 46–48 h after the
PMSG injection.
3. Prepare three IVF dishes: Place a 200-μl drop of HTF in each
dish and cover the medium with mineral oil.
4. Prepare one sperm preincubation dish: Place a 100-μl drop of
CARD FERTIUP Mouse Sperm Preincubation Medium and
cover the medium with mineral oil.
5. Prepare three Wash dishes: Place three 100-μl drops of KSOM
in each dish and cover the medium with mineral oil.
6. Prepare culture dishes: Place five 20-μl drops of KSOM in each
dish and cover the medium with mineral oil. The number of
culture dishes depends on the number of mouse zygotes.
50 zygotes can be cultured in one dish (10 zygotes in each
20-μl KSOM drop).
7. Collect sperm from the epididymis and transfer it into the
sperm preincubation dish. Incubate the dish for 1 h in a
37
C incubator with 5% CO 2 .
8. Collect oocyte masses from the oviducts of the female mice
13 h after hCG injection and place them in the IVF dishes.
Oocyte masses from two female mice are placed in one
dish. Incubate the dish for 30 min in a 37
C incubator with
5% CO 2 .
9. Transfer the sperm suspension into the IVF dish and incubate
the dish for 3–5 h in a 37
C incubator with 5% CO 2 .
10. Collect and wash the presumptive zygotes with KSOM
medium in the WASH dish by mouth pipette.
11. Move the zygotes into the culture dishes. Place 10 zygote in
one 20-μl KSOM drop. Incubate the dishes until
electroporation.
3.2 Genome Editing
Solution
1. Mix 0.6 μl of 1 μg/μl crRNA, 0.6 μl of 1 μg/μl tracrRNA, and
4.2 μl of Opti-MEM medium (5.4 μl total). For the
HDR-mediated knockin, add 0.6–1.2 μl of 1 μg/μl ssODN
to the mixture (see Note 4).
2. Heat the mixed reagents at 95
C for 3 min.
3. Add 0.6 μl of 1 μg/μl Cas9 into the mixed reagents (6 μl total).
4. Place the mixed reagents described above on ice until use.
Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically. . .
123