To perform proper animal experimentation, contact animal facility in your institute. Animal experiments must comply with national regulations concerning animals and their use.
Lentiviral vector mediated transgenesis5 should be performed as control experiment.
Preparation of blastocysts4 (Day 1-7)
1| Inject 5 units of PMSG intraperitoneally around noon. (Day 1)
2| Inject 5 units of HCG intraperitoneally after 48 hrs of interval. (Day 3)
3| Mate superovulated females with stud males (one female per male) after HCG injection. (Day 4)
Stud males can be used twice a week (once a week is preferable).
4| Check vaginal plugs in the next morning (Day 5).
5| Collect embryos at either stage, 2-4 cells (protocol A) or blastocyst (protocol B). Usually more embryos can be collected with protocol A. However, 2-days of culture in vitro slightly delays the embryo development.
(A) Collection of 2-4 cells stage embryos in afternoon. (Day 5)
(i) Prepare flushing needle filled with FHM.
(ii) Remove oviducts from mated females and keep them covered with mineral oil.
(iii) Place each oviduct in 20 micro-L drop of FHM covered with mineral oil.
(iv) Insert flushing needle into infundibulum and harvest embryos with FHM.
(v) Collect embryos with embryo-handling pipette and then wash 3 times by serially transferring embryos in 50 micro-L of kSOM drops covered with mineral oil.
(vi) Incubate embryos at 37 oC and 5% CO2 for 2 more days.
(B) Collection of blastocysts in morning. (Day 7)
(i) Remove uterus from mated females and keep them covered with mineral oil.
(ii) Cut off both ends and flush out uterus with 100 micro-L of FHM with P200 pipette into 60 mm dishes.
(iii) Collect embryos with embryo-handling pipette and then wash 3 times by serially transferring embryos in 50 micro-L of kSOM drops covered with mineral oil.
(iv) Incubate embryos at 37 oC and 5% CO2 until use.
Zonal removal in acidic Tyode’s solution4 (Day 7)
6| Prepare 20 micro-L drops of acidic tyrode’s solution on 60 mm dishes. FHM washing drops should be prepared on same dish. Cover with mineral oil.
7| In a drop of acidic Tyrodes’s solution, pipette blastocysts up and down 3-4 times in a few seconds and then transfer them into a next drop. After zona dissolves (around 30 seconds), wash zona-free blastocysts 3 times in FHM drops.
8| Wash 3 times in kSOM droplets and incubate at 37 oC and 5% CO2.
Lentiviral vector transduction1, 5, 6 (Day 7)
9| Thaw lentiviral vector stock solution and spin down (10,000 rpm for 5 sec) to remove debris.
10| Carefully transfer supernatant into a new tube, then dilute with kSOM (final concentration is 1 x 103 ng-p24/ml), and prepare 5-10 micro-L drops on 60 mm dishes. Cover with mineral oil.
11| Pipette up and down blastocysts in the first drop and then transfer each blastocyst in each drop.
12| Incubate them at 37 oC and 5% CO2 for 5 hours then wash 3 times in kSOM drops.
Transplantation and analysis4 (Day 7-24)
13| Transfer 8-10 transduced blastocysts into each uterus of pseudopregnant females (2.5 dpc).
14| Analyze at appropriate time during pregnancy. (Day 7-24)
15| Deliver pups and placentas by caesarian section in Day 24 morning (19.5 dpc). Normal delivery should be prevented by progesterone injection on Day 22 and 23.
This protocol can be completed in 24 days.
Day 1: Inject PMSG around noon (30 min)
Day 3: Inject HCG 48 hrs after PMSG and mating with stud males (30 min)
Day 4: Check vaginal plug in morning (30 min)
Day 4: Mate ICR females with vasectomized males in evening (30 min)
Day 5: Check vaginal plug in morning (30 min)
Day 5: Collect 2-4 cell stage embryos by flushing out oviduct in afternoon (1-2 hr)
Day 7: Remove zona pellucida (1 hr)
Day 7: Transduce zona-free blastocysts (5 hr)
Day 7: Transfer the transduced blastocysts into pseudopregnant ICR female (1-2 hr)
Analyze at appropriate time until delivery
Day 22: Inject 2mg of progesterone around noon (30 min)
Day 23: Inject 2 mg of progesterone around noon (30 min)
Day 24: Deliver E19.5 pups and placentas by caesarian section in morning (1-2 hr)