Assisted Reproduction Technologies

Options for the rederivation of mouse lines from external sources:

In Vitro Fertilization

The preferred method for rederivation is in vitro fertilization (IVF) utilizing frozen sperm. The day after the IVF procedure, fertilized embryos are washed and prepared for transfer into appropriate recipients. Pups are typically born around 19 days later. At 3 weeks of age, the PI receives tail biopsies for genotyping, which must be completed before the mice are transferred to the investigator’s room for housing.

Frozen Embryo Cryorecovery

Frozen embryos, typically at the 2- to 8-cell stage, are accepted. These embryos are thawed according to the protocol employed by the lab that cryopreserved them, thoroughly washed, and subsequently transferred into a recipient female. Pups are born approximately 19 days later. At 3 weeks of age, the PI receives tail biopsies for genotyping, a prerequisite before transferring the mice to the investigator’s room for housing and breeding.

Fresh Embryo Rederivation

Similar to frozen embryo rederivation, this approach requires close coordination between the lab sending the embryos and the Vanderbilt Genome Editing Resource to prepare for embryo transfer surgeries. Embryos, ranging from the 2-cell stage to the morula/blastocyst stage, can be shipped for transfer. Pups are typically born around 19 days after the embryo transfer. At 3 weeks of age, the PI receives tail biopsies for genotyping, which must be completed before transferring the mice to the investigator’s room for housing.

Options for the rederivation of mouse lines maintained at Vanderbilt:

In Vitro fertilization (IVF)

Fresh or frozen sperm from a transgenic male is used to fertilize oocytes obtained from superovulated female mice. IVF rederivations generally yield a larger number of pups compared to thawing one or two straws of embryos for rederivation. After overnight incubation and fertilization, two-cell embryos are transferred into recipient animals. Pups are born approximately 19 days later, and at 3 weeks of age, the Vanderbilt Genome Editing Resource staff performs weaning, tailing, and ear punching. The investigator then screens the tail DNA for transgenic founders and notifies the resource of the results.

Line Expansion by IVF

To establish a colony of experimental animals from a single founder, sperm from N1 heterozygous males is harvested and used to fertilize multiple isogenic wild-type embryos. Approximately half of the resulting N2 generation would be heterozygous, allowing for the production of a sufficient number of animals for experimental analysis in the first F1 generation. This approach potentially saves two generations of natural breeding time.

Embryo Transfer Rederivation

For rederivation, fresh or frozen embryos ranging from 0.5 to 3.5 days old are washed extensively in sterile medium and transferred into recipient females. Pups are born after seventeen to nineteen days, and at 3 weeks of age, the Vanderbilt Genome Editing Resource staff performs weaning, tailing, and ear punching. The investigator then screens the tail DNA for transgenic founders and notifies the resource of the results. This service is recommended for homozygous lines or lines with multiple genetic mutations.

Embryo Retrieval and Transfer Rederivation

Ovary Transplants

Additionally, ovary transplantation is available as a service when a valuable line of animals ceases to reproduce and is at risk. In this procedure, ovaries from a transgenic female are removed and transplanted into 1-2 recipient animals. Although not frequently utilized, this service is essential in preserving the line.