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Mutations in the well-known Myostatin (MSTN) produce a ‘double-muscle’ phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant ‘double-muscle’ phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca2+-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.

Abstract Background Alpha 1-antitrypsin (α1-AT) may affect the susceptibility of mastitis in dairy cattle for its possible role in the protection of lactoferrin from proteolytic degradation in the mammary. Milk somatic cell score (SCS) is a logarithmic transformation of the milk somatic cell count widely used as an index to evaluate mastitis. To study the relationships of α1-AT gene and SCS in Chinese Holstein cows, methods of PCR-SSCP, DNA sequencing, PCR-RFLP, and CRS-PCR technologies were used to detect single nucleotide polymorphisms sites in α1-AT gene. Results Two polymorphic sites at G5503A and G5746C of α1-AT gene were found. AA (0.3633), AB (0.4644) and BB (0.1723) genotypes were detected at G5503A site, CC (0.3483), CD (0.4906) and DD (0.1611) genotypes were found at G5746C in Chinese Holstein cows. Least squares mean of SCS for individuals with BB genotype was significantly lower than that with AA and AB genotype (p 0.05). Least squares mean of SCS for individuals with BBDD genotype combination were significantly lower than those with AACC and AACD (p