Preliminary Phenotypic Analysis of Myh13 Knockout Mouse

doi:10.3969/j.issn.1674-5817.2019.03.004

Abstract

Abstract: Objective A preliminary phenotypic analysis of the Myh13 knockout mouse model was performed in order to investigate the biological function of Myh13. Methods Expression of Myh13 in mice was determined by qRT-PCR and Western blotting. Validation of Myh13 knockout mouse was analyzed by gene sequencing, qRT-PCR and Western blotting. Genotypic ratio in the offsprings from the self-cross of heterozygotes was calculated. Changes in body weight and serum biochemistry parameters were measured. Histopathological features of mouse extraocular muscles were displayed by H&E staining and transmission electron microscopy examination. Results In mice, Myh13 is specifically expressed in extraocular muscle. It was verified that the target gene in Myh13 knockout mouse was successfully deleted at DNA, RNA and protein levels. Genotypic ratio of heterozygotes in the offsprings was consistent with Mendel’s law. No significant differences existed among different genotypes in body weight or serum biochemical analysis. HE staining revealed no remarkable change in homozygous and heterozygous mouse extraocular muscles compared to wild-type mouse extraocular muscles. Electron microscopy of mouse extraocular muscles showed that fat droplets deposited in heterozygotes and homozygotes. Sarcoplasmic reticulum dilation was also identified in homozygotes. Conclusions Knockout of Myh13 can alter the microstructure of extraocular muscles in mice, which may affect the function of extraocular muscles.

Key words: Myh13, Mouse model, Extraocular muscle

CLC Number:

Q95-33

HE Yi-min, GU Ming-min. Preliminary Phenotypic Analysis of Myh13 Knockout Mouse[J]. Laboratory Animal and Comparative Medicine, 2019, 39(3): 193-200.

References

[1] Foth BJ, Goedecke MC, Soldati D.New insights into myosin evolution and classification[J]. Proc Natl Acad Sci U S A, 2006, 103(10):3681-3686.
[2] Weiss A, Leinwand LA.The mammalian myosin heavy chain gene family[J]. Annu Rev Cell Dev Biol, 1996, 12(1):417-439.
[3] Clark KA, McElhinny AS, Beckerle MC, et al. Striated muscle cytoarchitecture: an intricate web of form and function[J]. Annu Rev Cell Dev Biol, 2002, 18(1):637-706.
[4] Pedrosa-Domellof F, Holmgren Y, Lucas CA, et al.Human extraocular muscles: unique pattern of myosin heavy chain expression during myotube formation[J]. Invest Ophthalmol Vis Sci, 2000, 41(7):1608-1616.
[5] Canepari M, Pellegrino MA, D'Antona G, et al. Skeletal muscle fibre diversity and the underlying mechanisms[J]. Acta Physiol (Oxf), 2010, 199(4):465-476.
[6] Bloemink MJ, Deacon JC, Resnicow DI, et al.The superfast human extraocular myosin is kinetically distinct from the fast skeletal IIa, IIb, and IId isoforms[J]. J Biol Chem, 2013, 288(38):27469-27479.
[7] Stuelsatz P, Yablonka-Reuveni Z.Isolation of mouse periocular tissue for histological and immunostaining analyses of the extraocular muscles and their satellite cells[J]. Methods Mol Biol, 2016, 1460(1): 101-127.
[8] Schachat F, Briggs MM.Phylogenetic implications of the superfast myosin in extraocular muscles[J]. J Exp Biol, 2002, 205(15):2189-2201.
[9] England J, Loughna S.Heavy and light roles: myosin in the morphogenesis of the heart[J]. Cell Mol Life Sci, 2013, 70(7):1221-1239.
[10] Carroll KJ, Makarewich CA, McAnally J, et al. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9[J]. Proc Natl Acad Sci U S A, 2016, 113(2):338-343.
[11] Teekakirikul P, Eminaga S, Toka O, et al.Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires TGF-beta[J]. J Clin Invest, 2010, 120(10):3520-3529.
[12] Eichel Y, Tormos LM, Squires JE.Preoperative use of platelets in a 6-year-old with acute appendicitis and a myosin heavy chain 9-related disorder: a case report and review of literature[J]. Transfusion, 2016, 56(2):349-353.
[13] Matsushita T, Hayashi H, Kunishima S, et al.Targeted disruption of mouse ortholog of the human MYH9 responsible for macrothrombocytopenia with different organ involvement: hematological, nephrological, and otological studies of heterozygous KO mice[J]. Biochem Biophys Res Commun, 2004, 325(4):1163-1171.
[14] Zhang Y, Conti MA, Malide D, et al.Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A[J]. Blood, 2012, 119(1):238-250.
[15] Altick AL, Feng CY, Schlauch K, et al.Differences in gene expression between strabismic and normal human extraocular muscles[J]. Invest Ophthalmol Vis Sci, 2012, 53(9): 5168-5177.
[16] 田亮, 邓大明, 申煌煊. 共同性内斜视患者眼外肌中肌球蛋白重链表达的研究[J]. 眼科新进展, 2011, 31(6):508-510.
[17] 罗琪, 周炼红, 易贝茜, 等. 共同性斜视眼外肌的病理变化及肝细胞生长因子表达研究[J]. 中华眼视光学与视觉科学杂志, 2015, 17(10):621-624,630.
[18] Papizan JB, Garry GA, Brezprozvannaya S, et al.Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice[J]. J Clin Invest, 2017, 127(10):3730-3740.
[19] Deguise MO, Boyer JG, McFall ER, et al. Differential induction of muscle atrophy pathways in two mouse models of spinal muscular atrophy[J]. Sci Rep, 2016, 6(1):28846-28860.