OHSU

Protocols

Embryo generation

Superovulation

Day Action Embryo Stage Project Types
Day 0 PMSG, 5 IU, IP

Day 1


Day 2 hCG, 5 IU, IP

Day 3 e0.5 single celled embryos Pronuclear injection
Day 4 e1.5 2-cell embryos  
Day 5 e2.5 morulae Aggregation, harvest for blastocyst injection
Day 6 e3.5 blastocysts Blastocyst injection


Harvest

3 to 5 week old mice are superovulated and checked for the presence of a copulation plug.  After cervical dislocation, fertilized embryos are collected by (e0.5) tearing the ampulla and releasing them into a hyaluronidase/M2 solution for dissociation, (e1.5-e2.5) flushing out via the infundibulum of the oviduct or (e3.5) flushing out of the uterus.  Embryos are maintained in gassed KSOM or M16 in a water jacketed, 5% CO2 incubator at 37°C and 95% humidity.

Surgeries

Implantation of injected embryos into pseudopregnant foster mother mice

Fur over the left lumbar area, approximately 2 cm2, is sprayed with 70% EtOH, and a 1 cm incision is made. After opening the abdominal cavity, the fat pad overlying the ovary is grasped with sterile forceps, and the ovary and distal end of the uterine horn is exteriorized. Embryos can either be transferred into the oviduct (zygotes, 2-cell embryos) or the uterus (blastocysts). For 2-cell embryos, the tip of the pipette is inserted into the infundibulum and with the application of positive air pressure, 10 to 20 embryos are placed into the fallopian tube. Transfer of blastocysts is achieved by generating a port at the distal end of the uterus using a hypodermic needle followed by transfer of 8 to 10 blastocysts into one horn of the uterus. Following embryo transfers, the abdominal wall is sutured with 3-0 or 4-0 PDS-II taper and the skin incision is closed using surgical staples or PDS-II. Post-surgical care includes a heating pad or lamp to avoid hypothermia until the mice are recovered from anesthesia.  

Vasectomy of Mice (males 6-8 weeks of age)

The abdomen is cleaned with 70% ethanol, and a 1.0 cm transverse incision is made in the ventro-distal abdomen. The fat pads overlying the testis and vas deferens are grasped and exteriorized using sterile forceps. Both vas deferentia are then cauterized, and the testes are replaced into the abdominal cavity. The incision is closed as described above. Post-surgical care includes a heating pad or lamp to avoid hypothermia until the mice are recovered from anesthesia. Animals are naturally mated or mated to superovulated females and a minimum of 2 plugged, non-pregnant females indicates a successful vasectomy.

Cell culture

ES cell culture

ES cells must be kept in R1 media with LIF. Media must be changed every day and cells need to be passaged well before reaching confluence. Failure to do so results in differentiation of stem cells and renders the culture unusable. 

Generating MEFs

Embryos are harvested from naturally mated or superovulated and mated females between e12.5 and e15.5 with the optimal time being e13.5. Heads and internal organs of the embryos are removed and the remaining tissue is rinsed in sterile PBS and minced. After the addition of trypsin and DNAseI the tissue is incubated for 15 minutes and then passed through a 16G needle. The resulting mixture is centrifuged at roughly 1000RPM for 5 minutes and the supernatant is collected and spun again and the very loose pellet of cells is plated in MEF media. The supernatant is allowed to incubate for another 10-15 minutes, spun down and the resulting loose pellet is plated.

Molecular procedures

DNA purification

We use the UltraClean GelSpin Mo Bio kit (catalog # 12400-50) to purify all DNA constructs prior to use in our projects.