Getting Around the Plug Check
Research Models
Rhonda Wiler

Getting Around the Plug Check

Ultrasound confirmation of pregnancy in genetically modified mice reduces resources and enhances reliability

Providing timed-pregnant, genetically-altered mice is a service the Transgenic Technology Department provides to investigators at Genentech. Until recently, the standard method we used to identify timed-pregnant females was the "plug check"—hardened seminal fluid that blocks the vagina and remains in place for about 12 hours after mating.

Unfortunately, due to variability in male fertility, approximately 40% of plugged females are not actually pregnant at the time of embryo collection. Not only does this set back our investigators' experimental timelines, it wastes difficult-to-obtain reagents like the complex mutant mice.

In a triple mutant colony that requires wild-type littermate controls, only 1 out of 16 animals produced will be females of the correct genotype for embryo harvest. This means that 15 animals have to be produced and culled in order to provide one donor female of the desired genotype. And if this female is not pregnant, 16 animals have actually been wasted.

Clearly, having the ability to confirm pregnancy prior to transfer and harvest would go a long way toward reducing animal waste and increasing the reliability of the service. Which brings us to ultrasound. Because this is the standard technique used in clinical settings to stage embryo development non-invasively we wanted to see if it also had utility in a high-throughput setting, in plugged female mice.

First, we performed a feasibility study to determine which type of ultrasound instrumentation was ideal for staging embryo development in mice and to assess any training and throughput challenges prior to committing resources. The findings from the feasibility study were promising so we leased a Visualsonics Vevo 770 imaging system with a RMV 704 scan-head. Next, we selected a team of technicians from the Transgenic Technology Department to prototype and scale the new process. They succeeded in developing a high volume, ultrasound imaging process that can be performed with a high degree of accuracy.

The imaging process takes about three minutes per animal. The new service was offered to a limited number of investigators to validate and then rolled out as a standard service to all our investigators who use genetically modified animal models.

The results speak for themselves. Implementation of this procedure significantly reduces the number of animals that are wasted due to the transfer of non-pregnant females. By looking at the breeding scheme that produced the females used in the timed pregnancies, we determined that 5.2 animals of an undesired genotype or gender must be produced for each female transferred. When we include the non-pregnant female, we determined that 6.2 animals are wasted when a non-pregnant female is transferred and used experimentally.

So by performing ultrasound, these non-pregnant females can now be detected prior to shipment and then returned to the colony for re-use. In a four-year period, we have performed 10,788 scans after setting up over 18,000 females in timed-pregnant matings. We found an overall plug rate of 59% and a pregnancy rate of 64% among the plugged animals. Using ultrasound to detect pregnancy resulted in a reduction of animal use by nearly 42,000 mice to date.

That's pretty sound science.

General procedure for performing ultrasound for pregnancy confirmation and for performing embryo counting.

  1. Prepare for ultrasound by anesthesizing the animal followed by the chemical removal of the abdominal furUltrasound of bladder
  2. Place and secure the animal on ultrasound staging platform
  3. Apply ultrasound gel onto abdomen of the animal and lower ultrasound probe to begin scanning
  4. Locate and center the animal's bladder on the monitor
  5. Ultrasound scan consists of starting from the animal's bladder and ending at the left uterine horn. Repeat the process for the right uterine horn and record the total embryo count (shown in images at right).
  6. Remove the animal from the staging platform when the scan is complete. Place the animal into warmed recovery cage for post-procedure monitoring (shown at left).. Mice being monitored in recovery cage

Images courtesy of GenentechUltrasound of embryos