LMNA-mutated Rabbits: A Model of Premature Aging Syndrome with Muscular Dystrophy and Dilated Cardiomyopathy
Tingting Sui1, Di Liu1, Tingjun Liu1, Jichao Deng1, Mao Chen1, Yuanyuan Xu1, Yuning Song1, Hongsheng Ouyang1, Liangxue Lai1,2,*, Zhanjun Li1,*
1Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, Changchun 130062, China 2Key Laboratory of Regenerative Biology, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China
Premature aging syndromes are rare genetic disorders mimicking clinical and molecular features of aging. Products of the LMNA gene, primarily lamin A and C, are major components of the nuclear lamina. A recently identified group of premature aging syndromes was related to mutations of the LMNA gene. Although LMNA disorders have been identified in premature aging syndromes, affect specifically the skeletal muscles, cardiac muscles, and lipodystrophy, understanding the pathogenic mechanisms still need to be elucidated. Here, to establish a rabbit knockout (KO) model of premature aging syndromes, we performed precise LMNA targeting in rabbits via co-injection of Cas9/sgRNA mRNA into zygotes. The LMNA-KO rabbits exhibited reduced locomotion activity with abnormal stiff walking posture and a shortened stature, all of them died within 22 days. In addition, cardiomyopathy, muscular dystrophy, bone and joint abnormalities, as well as lipodystrophy were observed in LMNA-KO rabbits. In conclusion, the novel rabbit LMNA-KO model, displayed typical features of histopathological defects that are observed in premature aging syndromes, and may be utilized as a valuable resource for understanding the pathophysiological mechanisms of premature aging syndromes and elucidating mysteries of the normal process of aging in humans.
Figure 1. Generation of LMNA-KO rabbits using CRISPR/Cas9 system. (A) Schematic diagram of the two sgRNA target sites located in exon 3 of the rabbit LMNA locus. LMNA exons are indicated by pink rectangles; target sites of the two sgRNA sequences, sgRNA1 and sgRNA2, are highlighted in red; protospacer-adjacent motif (PAM) sequence is highlighted in green (B) Mutation detection by T7E1 cleavage assay in rabbit pups 1-15. Gel images have been cropped. M, DL2000, has used to indicate band size. (C) Mutation detection by T7E1 cleavage assay in rabbit pups (n=16-32 pups). Gel images have been cropped. M, DL2000, has been used to indicate band size. Black line indicates the WT allele (490 bp). (D) T-cloning and Sanger sequencing of modified LMNA alleles in 1-15 pups. WT sequence is shown at the top of the targeting sequence. PAM sites are highlighted in green; target sequences are shown in red; deletions (-); insertions are shown in blue; WT, wild-type control.
Blastocyst with Mutantion (%)
Table 1. Summary of embryo microinjections of Cas9/sgRNA in zygotes.
Figure 2. Phenotype characterization of LMNA-KO rabbits. (A) The gross performance of 14-day-old LMNA-KO rabbits by photo, and joint stiffness (white arrows) by X-ray autoradiography examination. (B) Hind legs of WT and LMNA-KO rabbits showing stiff ankle joints in LMNA-KO rabbits (Red oval). (C) X-ray absorptiometry of hind legs from WT and LMNA-KO rabbits showing stiff ankle joints in LMNA-KO rabbits (Red oval). (D) H&E-staining of the skin, LMNA-KO rabbit showed decreased eccrine in skin (E) Behavioral photographs cut from video of the LMNA-KO-1, LMNA-KO-2, and WT control. (F) Body-weight comparison of LMNA-KO and WT rabbits from newborn to 22 days. (G) Survival curves of LMNA-KO and WT rabbits. Scale bar, 50 μm.
Figure 3. Cardiomyopathy of LMNA-KO rabbits. (A) The heart from 18-day-old LMNA-KO rabbits and WT control. (B) The increased left ventricular diastolic diameter in LMNA-KO rabbits. (C) The decreased left ventricular ejection fraction (EF) in LMNA-KO rabbits. (D) The decreased fractional shortening in LMNA-KO rabbits. (E) The decreased heart rate in LMNA-KO rabbits. Data were presented as means ± SEM of at least three rabbits per group and analyzed by Student’s t-tests using GraphPad Prism software 7.0. * p < 0.05; ** p < 0.01; *** p < 0.001. Normalized LVDD, LV diastolic diameter-to-body weight ratio. (F) H&E-staining and Masson’ trichrome-staining of cardiac muscles from WT and LMNA-KO rabbits, showing significant fibrosis (Red arrows) and cardiomyocytes loss (Blue arrows) in LMNA-KO rabbits. (G) H&E-staining and Oil red-staining of the cardiac muscles from WT and LMNA-KO rabbits, showing significant fat infiltration (Green arrows) in LMNA-KO rabbits. (H) Aorta tissues of the 18-day-old LMNA-KO rabbits and WT controls. (I) H&E-staining of the aorta from WT and LMNA-KO rabbits. Scale bar, 50 μm.
Pups obtained (%transferred)
Pups with mutations (% pups)
Pups with biallelic mutations (%pups)
Table 2 Generation of LMNA-KO rabbits using CRISPR/Cas9 system.
Figure 4. Muscular dystrophy in LMNA-KO rabbits. (A) Gross muscles of the 18-day-old LMNA-KO rabbits and WT control by photo. (B) Masson’s trichrome staining of the tongue, bladder and diaphragm muscles from WT and LMNA-KO rabbits, showing inflammatory cells infiltration in the tongue (Green arrows), and thinner bladder muscles of LMNA-KO rabbits (Black arrows). (C) Longitudinal section of the H&E-staining and Masson’s trichrome staining of the gastrocnemius muscles from WT and LMNA-KO rabbits, showing inflammatory cells infiltration (Green arrows) in LMNA-KO rabbits. (D) Cross section of the H&E-staining and Masson’s trichrome stained of the gastrocnemius muscles from WT and LMNA-KO rabbits, showing a wide variation in the fiber size (Black arrows), an increased number of atrophic fibers, hypertrophic fibers and lobulated fiber (Blue arrows) in LMNA-KO rabbits. (E) Statistical analysis of the mean fibers diameter of the muscle fibers from the gastrocnemius muscles of the 18-day-old WT and LMNA-KO rabbits. (F) Statistical analysis of the mean fibers area of the muscle fibers from the gastrocnemius muscles of the 18-day-old WT and LMNA-KO rabbits. Scale bar, 50 μm.
Figure 5. Bone abnormalities of LMNA-KO rabbits. X-ray absorptiometry of the femur (A) and the tibia (B) from WT and LMNA-KO rabbits. Statistical analysis of the average femur length (C) and tibia length (D) in WT and LMNA-KO rabbits. Data are presented as means ± SEM of at least three rabbits per group and analyzed by Student’s t-test using Graphpad Prism software 7.0. * p < 0.05; ** p < 0.01; *** p < 0.001. (E) H&E-staining of the cortical bone and diaphysis bone from WT and LMNA-KO rabbits, showing decreased cortical bone width, significantly reduced numbers of the osteoblasts and osteocytes in LMNA-KO rabbits. (F) H&E-staining of the wrist and growth plates from WT and LMNA-KO rabbits, showing rough articular surface and irregular arrangement of the growth plate with more porous areas in LMNA-KO rabbits. Scale bar, 50 μm.
Figure 6. Lipodystrophy of LMNA-KO rabbits. (A) Dysplasia of the adipose tissue in LMNA-KO rabbits by anatomy analysis. (B) The liver of LMNA-KO rabbits and WT controls. (C) H&E-staining and Oil red staining of the brown adipose tissue from LMNA-KO rabbits and WT rabbits. (D) H&E-staining of liver sections from WT and LMNA-KO rabbits. (E) Gene expression of PPARɡ, SREBF1, GLUT4, FABP4, and ADIPOQ was determined by qRT-PCR. WT, WT control; LMNA-KO, LMNA gene knockout rabbit. All experiments were repeated for three times for each gene. Data are presented as the mean ± SEM and analyzed by t-tests using Graphpad Prism software 6.0. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 50 μm.
T. Chol (mmol/l)
1.90 ± 0.37
7.77 ± 0.39a
2.72 ± 0.25
2.37 ± 0.20
1.35 ± 0.12a
1.97 ± 0.23
1.37 ± 0.05a
0.71 ± 0.07
1.26 ± 0.31
4.66 ± 0.56a
1.37 ± 0.15
4.94 ± 0.44a
Table 3 Serum biochemical analysis in WT and LMNA-KO rabbits.
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