Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation / 生物工程学报

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- 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 MSTN 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 MSTN 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-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 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) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- 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. MSTN-/- 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.

Construction of point mutation rabbits using CRISPR/Cas9 / 浙江大学学报·医学版

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.

Correlation between serum miR-320b and carotid atherosclerosis in patients with acute ischemic stroke / 国际脑血管病杂志

Objective To investigate the correlation between serum miR-320b and carotid atherosclerosis in patients with acute ischemic stroke.Methods From January 2017 to December 2017,patients with acute ischemic stroke visited the Department of Neurology,the Affiliated Hospital of Yangzhou University were enrolled.According to the findings of carotid artery ultrasonography,they were divided into plaque group and plaque-free group.The baseline clinical data such as demographic data,vascular risk factors,and blood biochemical indicators were collected.Reverse transcription quantitative polymerase chain reaction was used to detect the expression level of serum miR-320b.Multivariatelogistic regression analysis was used to determine the independent risk factors for carotid atherosclerosis.Results A total of 135 patients with acute ischemic stroke were enrolled in this study,including 58 females and 77 males,aged 58.4 ± 10.6 years.There were 85 patients in the plaque group and 50 in the plaque-free group.The total cholesterol (t =5.523,P =0.023) and low-density lipoprotein cholesterol (t =4.415,P =0.044) in the plaque group were significantly higher than those in the plaque-free group,while high-density lipoprotein cholesterol (t =5.849,P=0.017) and serum miR-320b (t =4.331,P=0.039) were significantly lower than those in the plaque-free group.Multivariate logistic regression analysis showed that referring to the highest quartile group,the low serum miR-320b level might be an independent risk factor for carotid atherosclerosis (the first quartile group:odds ratio 2.701,95％ confidence interval 1.154-6.321,P =0.022;the second quartile group:odds ratio 2.521,95％ confidence interval 1.249-5.091,P =0.010;and the third quartile group:odds ratio 1.849,95％ confidence interval 1.041-3.283,P=0.036).Conclusion The low serum miR-320b level might be an independent risk factor for carotid atherosclerosis in patients with acute ischemic stroke.