[Propagation and phenotypic analysis of mutant rabbits with homozygous mutation].

Myostatin gene () encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-: 3; M2-: 2; M3-: 2; M7-: 6; M7-: 2). The body weight of homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of wild-type rabbits of the same age ((2 718±120) g . (1 969±53) g, < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm . (1 274.8±327.3) μm, < 0.01), but also significantly higher than that of hemizygous rabbits ((3 512.2±439.2) μm . (2 610.4±604.4) μm, < 0.05). In summary, five homozygous mutants rabbits of gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.