A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia
- State Key Laboratory of Animal Biotech Breeding, Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, China
- Institute of Animal Husbandry and Veterinary Medicine, Tianjin Academy of Agricultural Sciences, China
- National Center of Technology Innovation for animal model, NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, China
- College of Pulmonary and Critical Care Medicine, Chinese PLA General Hospital, China
Figures
Figure 1
Efficient generation of sheep carrying biallelic mutations in dual gene via the CRISPR/Cas9 system.
(A) Schematic of sgRNAs specific to exon 3 of the sheep MSTN locus. The crRNA sequences are highlighted in blue typeface and the PAM in red. (B) Schematic of sgRNAs specific to exon 3 of the sheep …
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Figure 1—source data 1
Uncropped and labeled gels for Figure 1C.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig1-data1-v1.zip
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Figure 1—source data 2
Raw unedited gels for Figure 1C.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig1-data2-v1.zip
Figure 2 with 2 supplements
The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant ‘double-muscle’ phenotype and muscle fiber hyperplasia.
(A) The 6-month-old WT and MF-/- sheep. The genome-edited sheep displayed an obvious ‘double-muscle’ phenotype compared with the WT. (B) The CT scanning image of the brisket and hip of WT and MF-/- …
Figure 2—figure supplement 1
FGF5 mutation does not affect muscle fiber size.
(A) HE sections of gluteus medius in WT and FGF5+/- sheep. Scale bar +/- μm. (B) Quantification of muscle fibre cell area of gluteus medius in WT and FGF5+/- sheep. (C) Quantification of muscle …
Figure 2—figure supplement 2
The MSTNDel73C mutation with FGF5 knockout has no potential effect on MSTN expression and muscular dystrophy.
(A) The mRNA expression levels of MSTN and FGF5 of gluteus medius in WT (n=4) and MF+/- (n=4) sheep at 3-month-old. (B) MSTN mRNA expression level of gluteus medius in WT (n=3) and MF+/- (n=4) …
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Figure 2—figure supplement 2—source data 1
Uncropped and labeled blots for Figure 2—figure supplement 2C.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Raw unedited blots for Figure 2—figure supplement 2C.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig2-figsupp2-data2-v1.zip
Figure 3 with 1 supplement
The MSTNDel73C mutation with FGF5 knockout promote proliferation and inhibit differentiation of skeletal muscle satellite cells.
(A) The number of cells was detected by CCK-8 at 12 hr, 24 hr, 36 hr, 48 hr, and 60 hr in GM (n=7–8 per group). (B–C) EdU assay showed that the number of EdU positive cells and EdU labeling index …
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Figure 3—source data 1
Uncropped and labeled blots for Figure 3H.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig3-data1-v1.zip
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Figure 3—source data 2
Raw unedited blots for Figure 3H.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig3-data2-v1.zip
Figure 3—figure supplement 1
The myogenic differentiation ability of MF+/- cells was continuously inhibited.
(A) The MyoG and MyHC immunofluorescence staining of myotubes at DM4. Scale bar 130 μm. (B–E) The myotube fusion index, number of myotubes, number of nuclei per myotube and the myotube diameter at …
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Figure 3—figure supplement 1—source data 1
Uncropped and labeled blots for Figure 3—figure supplement 1K.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Raw unedited blots for Figure 3—figure supplement 1K.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
The MSTNDel73C mutation with FGF5 knockout contributes to muscle phenotype via MEK-ERK-FOSL1 axis.
(A) Go enrichment analysis of DEGs. Among them, the top 20 entries with significant enrichment are listed in biological process (BP). CC, cellular component; MF, molecular function. (B) KEGG …
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Figure 4—source data 1
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig4-data1-v1.zip
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Figure 4—source data 2
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig4-data2-v1.zip
Figure 4—figure supplement 1
The effects of MSTN signaling pathway and MF+/- serum on proliferation and differentiation of skeletal muscle satellite cells.
(A) The mRNA expression levels of type I and type II receptors of MSTN between WT (n=3) and MF+/- (n=4) sheep gluteus medius. (B) The mRNA expression levels of the Smad family downstream of MSTN …
Figure 5
The overexpression of FOSL1 promotes proliferation and inhibits differentiation of skeletal muscle satellite cells.
(A) The mRNA expression level of FOSL1 at GM and DM2 after lentivirus infection (n=3). (B) The number of cells detected by CCK-8 at 0 hr, 12 hr, 24 hr, 36 hr, 48 hr, and 60 hr after infection with …
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Figure 5—source data 1
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig5-data1-v1.zip
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Figure 5—source data 2
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig5-data2-v1.zip
Figure 6
The inhibition of FOSL1 suppresses proliferation and promotes differentiation of skeletal muscle satellite cells.
(A) The mRNA expression level of FOSL1 at GM and DM2 after inhibiting FOSL1 (n=3). (B) The number of cells detected by CCK-8 at 0 hr, 12 hr, 24 hr, 36 hr, 48 hr, and 60 hr after inhibiting FOSL1 …
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Figure 6—source data 1
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig6-data1-v1.zip
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Figure 6—source data 2
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig6-data2-v1.zip
Figure 7
FOSL1 activity is a key regulator of myogenic differentiation and muscle myofiber hyperplasia.
(A) Immunofluorescence staining of myogenic differentiation markers MyoG and MyHC in sheep skeletal muscle satellite cells at DM2 after addition of 20 μM TBHQ. Scale bar 130 μm or 50 μm. (B–E) The …
Figure 8
The MSTNDel73C mutation with FGF5 knockout inhibit calcium-dependent transcription signal pathway.
(A–B) The protein expression level of CaMKII α/δ between WT and MF+/- cells at GM (n=3). (C) Distribution of intracellular Ca2+ signals between WT and MF+/- cells at GM. (D) Average intracellular Ca2…
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Figure 8—source data 1
Uncropped and labeled blots for Figure 8A.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig8-data1-v1.zip
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Figure 8—source data 2
Raw unedited blots for Figure 8A.
- https://cdn.elifesciences.org/articles/86827/elife-86827-fig8-data2-v1.zip
Figure 9
Schematic illustration of the regulation of muscle phenotypes by MSTNDel73C mutation with FGF5 knockout.
The MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic …
Additional files
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Supplementary file 1
Production and slaughter information of MSTNDel73C mutation with FGF5 knockout sheep.
(A) Summary of generation of sheep carrying biallelic mutations in dual genes via the CRISPR/Cas9 system. (B) Muscle weight of different parts in WT and MF+/- sheep (g). (C) The slaughter traits of muscles in WT and MF+/- sheep. (D) Meat quality of longissimus dorsi in WT and MF+/- sheep. (E) Shearing force of different parts in WT and MF+/- sheep (N). (F) Amino acid content of longissimus dorsi in WT and MF+/- sheep (%, DM basis).
- https://cdn.elifesciences.org/articles/86827/elife-86827-supp1-v1.docx
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Supplementary file 2
The information of primers sequence, siRNA sequence and antibody.
(A) Primers sequences of gene cloning. (B) The sequences of siRNA. (C) All primers of PCR and RT-qPCR. (D) The antibodies information.
- https://cdn.elifesciences.org/articles/86827/elife-86827-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/86827/elife-86827-mdarchecklist1-v1.docx
