Serving 435 principal investigators and 1,000-plus research protocols on campus, DLAM operates three tier-one animal facilities including a just-opened 17,000-ventilated cage vivarium—all “negative for all known viruses, bacteria, and parasites,” according to Marcelo Couto, DVM, campus veterinarian and DLAM executive director. New mouse strains are usually ordered from a handful of verified vendors, deemed “trusted sources,” which supply certified virus-free animals that can directly enter all vivaria, including the tier-one facility. However, not all desired mouse varieties are available through the trusted network. Obtaining the requested mice from other sources requires an eight-to-10 week quarantine before they are permitted to cross the barrier into a tier-two facility.

DLAM’s experience with quarantine showed it to be less than reliable in ruling out all disease.

“Quarantine can only test for known pathogens and it occasionally misses important organisms such as the ubiquitous mouse parvovirus (MPV) and as yet undiscovered organisms,” Couto points out. Other less common but serious pathogens, such as lymphocytic choriomeningitis virus (LCMV) and tropical rat mite can also be missed by traditional quarantine practices—both of these agents can affect people as well as mice.

“Furthermore, animals can still become contaminated in transit,” he continues. “Even when we test them on arrival and at the end of the quarantine, some mice may not develop adequate antibody titers that can be detected, so infection can be missed. As such, quarantine is not guaranteed to produce a clean mouse as its end product. Given the information we have accumulated over the years about age- and strain-dependence as well as seasonality of MPV infections, we concluded that traditional quarantine is ineffective and have therefore decided to stop the practice.”

Rederivation

Rederivation by embryo transfer is a surgical procedure that “cleans up” an infected mouse line to generate clean (i.e., uninfected) pups. As opposed to quarantine, rederivation all but guarantees a clean mouse as the end product. Essentially, rederivation acts like a buffer, inserting a generation of clean offspring between the imported mouse and its descendents, which lowers or eliminates the possibility of disease. The surgical embryo transfer gives UCLA control over the reproductive process even before fertilization assuring that the offspring will meet the clean criteria for either breeding or experimentation.

The process entails importing “dirty” (i.e., infected) males with the sought-after genetic mutation and then pairing them with clean females of the same “garden-variety” background, that is, the same strain but without the genetic manipulation. The egg-donor females are superovulated with sex hormones that more than double egg production (25 to 50 ovulated eggs instead of the typical 10 to 12) to bolster the chances of successful breeding. Mating takes place in the ART lab located outside the tier-one barrier facility.

“By mating dirty males to clean females, we create additional clean barriers, minimizing the chances that the embryos will be infected,” Couto says.

The next morning the female reproductive tracts are harvested in the dirty facility and then transported to a clean lab within the barrier. Here the embryos are washed extensively and implanted in pristine females with reproductive cycles synchronized to those of the egg donors. On waking from anesthesia, the foster-mom mice are pregnant and will give birth 19 to 21 days later.

Noting that one segment of the ART lab is considered contaminated because it receives the imported males and collects the egg-donor reproductive tracts, Couto emphasizes the importance of keeping the clean functions (embryo collection, washing, and implantation surgery) in distinct locations physically separated from each other.

“The foster mom and the egg donor are both clean, so chances of transmitting a virus to the offspring are remote,” he also points out.

The resulting clean babies will be used to start propagation of the particular mouse line in the tier-one facility. A small number of breeding stock is kept in the tier-one breeding facilities to be sure the line remains clean and uninfected.

“The breeding colony compartments are entirely controlled by our staff, with no researcher access,” explains Couto. “We supply investigators with breeding records and will increase or decrease the breeding rate of certain lines according to their research needs. Each compartment has its own health reporting system and sentinel program. If you are going through the trouble and expense of rederiving, it only makes sense to move in the direction of ‘super clean,’” he advises.

Administrative Buy-In

The success of DLAM’s rederivation program is borne out by the multi-year disease-free track records of the two older tier-one animal facilities. Couto is quick to credit the UCLA administration for funding the enabling ART lab, but he himself crafted a winning case for the new strategy. Invoking the economics, he proposed that the $1,500 to $1,600 cost for eight weeks of quarantine—which would not totally eliminate the risk of outbreak and re-population—failed to offer enough of a savings when compared to the guaranteed results of a $2,000 rederivation procedure.

“If after quarantine the animal turns out to have a disease, the time and money spent will have been wasted, and the whole process will have to start over,” he points out, adding, “The better we become at rederivation, with higher throughput and so on, the less expensive it will be in the long run. Quarantine is a fixed price, which will only go up with the increase in the cost of reagents, per diem charges, etc.”

He also persuaded the administration to subsidize the cost of rederivation, but not quarantine, in order to hasten its adoption by investigators.

“That way, they get all the benefits and none of the cost,” he explains. “Investigators who want to import mice from another institution may do so, but quarantine is mandatory, and the PI is billed for it. Rederivation is available at no charge, so the choice is not that difficult given the guaranteed results.”

Cryopreservation

Cryopreservation is another powerful population management technique in force at DLAM, allowing just-in-time mouse breeding to ease the persistent housing crunch.

“Even with very good long-range plans that call for new vivariums, construction invariably falls behind schedule and mouse populations continue to grow at an accelerated pace, clearly outstripping available capacity,” Couto remarks.

Investigators who will not need a particular strain for six months or longer are encouraged to have the embryos and sperm frozen and held in liquid nitrogen until it’s time for the next research phase. It takes three to four weeks to generate these animals-to-order—defrosting the embryos, preparing the foster mother, and so on—but at $100 per year for frozen storage, it costs much less than the $0.75 per day it takes to sustain a live animal.

As a solution, cryopreservation goes beyond cost and space issues, serving to counteract the accumulation of genetic mutations due to the rapid rate of mouse reproduction.

“The more times a mouse ovulates, the more likely a genetic error will occur, and over time the strain changes. By freezing embryos we arrest this genetic drift,” he points out.

The crowning advantage is the protection cryopreservation offers against catastrophic loss, such as earthquakes or floods.

“In the case of a flood, for example, frozen embryos don’t drown,” Couto quips, adding, “It’s also a good way to maintain a particular strain for sharing with other institutions for collaborations.”

Three Tiers

When a researcher needs animals for an experiment, mice or breeding pairs are transferred out of the tier-one barrier into a tier-two environment. It’s a one-way path with absolutely no possibility of return. The experimental vivarium is generally free of all viruses and bacteria, negative for parvovirus, mouse hepatitis, pinworms, and other common bugs, with the exception of helicobacter. Fortunately, this very prevalent pathogen affects only a small range of research, so it is not an overwhelming concern for most PIs, Couto points out.

DLAM provides husbandry and health check services to the experimental colonies, but unlike the tier-one facilities, researchers have ready access to these areas, with few restrictions on the procedures that can be done there. However, Couto anticipates the need for another kind of intermediate space outside the barrier, a third tier. The idea is to provide interim housing for imported mouse strains being evaluated for rederivation and further propagation. This approach will give PIs the opportunity to conduct limited breeding, blood testing, and other short-term research before establishing a lifetime colony.

“Under these circumstances there is no sense in going through rederivation or quarantine,” he advises. “At the end, if the researcher decides she doesn’t need or want the mouse, it would be a waste of money, time, and effort. If the strain is selected for breeding, the animals can be transferred into the rederivation program.”

Physically isolated from the tier-one barrier, the tier-three facility would also be serviced by DLAM and off-limits to researchers. UCLA’s DLAM currently runs a tier-three room, which allows for limited breeding and experimental manipulations of mice held short term.

“The real challenge will be to replicate functional areas, such as surgery, imaging, behavioral testing, and other specialized procedures within the tier-three facility and provide the DLAM staff with the necessary training to run this facility without (or with limited) researchers’ participation,” says Couto. “PIs traveling between tier-three and tier-two facilities could possibly introduce contaminants, so we would have to restrict access. This concept of a large, dedicated, tier-three vivarium is not quite a reality yet at UCLA, but we are already working on this idea on a limited scale.”

Streamlined Reporting

DLAM has also streamlined regulatory compliance by issuing Palm handheld devices with automated reporting functions to the appropriate animal facility staff. One program (SPECTOR) manages data collected through the soiled bedding sentinel system, transferring the information to a Web-based tool which then sends it directly to the pathologist and others in charge of the effort.

The other program (REACTOR) manages health tracking, allowing technicians to record details as they walk around the vivarium with their Palm devices. Drop-down menus minimize input errors. The devices can be synchronized wirelessly or from the desktop to send information to the veterinarian for review and appropriate action. A macro system also communicates problems to investigators, giving them treatment options via pull-down menus. Both programs and the macro system were developed in-house by the departmental IT manager.

“The investigators get the reports within minutes, and they need to respond by the deadline the veterinarian establishes, according to Institutional Animal Care Committee guidelines,” Couto explains. “This way we have a permanent electronic record of when a condition was found, reported to the vet, what the decision was, how it was communicated to the PI, what the response was, and the final disposition of the case. That’s how we close the loop.”

By Nicole Zaro Stahl