Refinements for embryo implantation surgery in the mouse: comparison of injectable and inhalant anesthesias - tribromoethanol, ketamine and isoflurane - on pregnancy and pup survival.

An essential aspect of genetically-engineered mice (GEM) is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudopregnant females for gestation, parturition, and growth to the weaning stage. This study was carried out to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tribromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or ketamine:medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used might affect the number of animals produced, each anesthetic agent was tested in implant surgeries and the numbers of pups produced using both wild-type and GEM embryos were assessed. Parallel studies were conducted in institutions in the EU and in the USA. Based on a direct comparison of pregnancy status, number of pups born, and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.

Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.

Cryopreserved morulae can be used to efficiently generate germline-transmitting chimeras by blastocyst injection.

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30-75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.