编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长
收稿日期: 2020-07-15
网络出版日期: 2021-02-03
基金资助
国家转基因生物新品种培育重大专项(2016ZX08006003-006);广东省重点领域研发计划项目(2018B020203003)
Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs
Received date: 2020-07-15
Online published: 2021-02-03
Supported by
the National Transgenic Major Program(2016ZX08006003-006);the Key R&D Program of Guangdong Province(2018B020203003)
肌生长抑制素(myostatin, MSTN)是转化生长因子β (transforming growth factor-β, TGF-β)家族成员之一,是一种肌肉生长抑制因子。解除MSTN的生长抑制功能是提高畜禽肌肉产量的一种有效途径。TGF-β的半胱氨酸节结构基元(cystine knot motif) 能够稳定MSTN蛋白结构,对MSTN生物学功能的发挥具有重要调控作用。本研究应用CRISRP/Cas9基因编辑技术在两广小花猪肾细胞(Liang Guang small spotted pig kidney cells, LPKCs)中对MSTN基因外显子3进行编辑,破坏了其半胱氨酸节基元,以解除MSTN对靶基因的抑制功能。将流式分选获得的混合阳性MSTN编辑LPKCs作为供体细胞进行核移植和胚胎移植,获得8头MSTN基因编辑两广小花猪仔猪,其中2头存活至10日龄,经鉴定这2头均为基因编辑杂合子,它们在构成MSNT蛋白半胱氨酸节基元的两个半胱氨残基C106和C108编码序列附近分别发生碱基的缺失与替换,导致移码突变,使C106和C108突变为其他氨基酸。MSTN基因编辑两广小花猪杂合子肩部和臀部肌肉较为发达。H&E切片分析显示,MSTN基因编辑猪肌纤维横截面积显著减少,肌纤维数量显著增多。Western Blot分析结果显示,C106和C108缺失对MSTN蛋白表达无显著性影响,但显著促进其靶基因Myf5、MyoD和Myogenin等成肌相关因子的表达。本研究获得的基因编辑猪模型没有造成MSTN表达完全缺失,可保留MSTN其他生物学功能,在促进两广小花猪肌肉生长的同时还消除了MSTN完全缺失可能对小花猪造成的潜在影响。
关键词： MSTN; CRISPR/Cas9; 两广小花猪; 基因编辑
彭定威, 李瑞强, 曾武, 王敏, 石翾, 曾检华, 刘小红, 陈瑶生, 何祖勇 . 编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长[J]. 遗传, 2021 , 43(3) : 261 -270 . DOI: 10.16288/j.yczz.20-222
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-β can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.
Key words： MSTN; CRISPR/Cas9; Liang Guang Small Spotted pig; gene editing
| [1] | WalkerRG, PoggioliT, KatsimpardiL, BuchananSM, OhJ, WattrusS, HeideckerB, FongYW, RubinLL, GanzP, ThompsonTB, WagersAJ, LeeRT. Biochemistry and biology of GDF11 and myostatin: similarities, differences, and questions for future investigation. Circ Res, 2016, 118(7): 1125- 1142. |
| [2] | McPherronAC, LawlerAM, LeeSJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 1997,387(6628):83- 90. |
| [3] | GrobetL, MartinLJ, PonceletD, PirottinD, BrouwersB, RiquetJ, SchoeberleinA, DunnerS, MénissierF, MassabandaJ, FriesR, HansetR, GeorgesM. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet, 1997, 17(1): 71- 74. |
| [4] | ClopA, MarcqF, TakedaH, PirottinD, TordoirX, BibéB, BouixJ, CaimentF, ElsenJM, EychenneF, LarzulC, LavilleE, MeishF, MilenkovicD, TobinJ, CharlierC, GeorgesM. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet, 2006, 38(7): 813- 818. |
| [5] | MosherDS, QuignonP, BustamanteCD, SutterNB, MellershCS, ParkerHG, OstranderEA. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genet, 2007,3(5): e79. |
| [6] | CatipovićB. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med, 2004, 351(10): 1030- 1031. |
| [7] | LeeSJ, McPherronAC. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA, 2001,98(16):9306- 9311. |
| [8] | SonstegardTS, RohrerGA, SmithTP. Myostatin maps to porcine chromosome 15 by linkage and physical analyses. Anim Genet, 1998, 29(1): 19- 22. |
| [9] | ThiesRS, ChenT, DaviesMV, TomkinsonKN, PearsonAA, ShakeyQA, WolfmanNM. GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF- 8 receptor binding. Growth Factors, 2001,18(4):251- 259. |
| [10] | WolfmanMW, McPherronAC, PappanoWN, DaviesMV, SongK, TomkinsonKN, WrightJF, ZhaoL, SebaldSM, GreenspanDS, LeeSJ. Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA, 2003,100(26):15842- 15846. |
| [11] | LeeSJ. Regulstion of muscle mass by myostatin. Annu Rev Cell Dev Biol, 2004, 20(1): 61- 86. |
| [12] | KambadurR, SharmaM, SmithTP, BassJJ. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res, 1997,7(9):910- 916. |
| [13] | StarckCS, Sutherland-SmithAJ. The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability. BMC Res Notes. 2011,4:442. |
| [14] | WangM, HuangX, ShiX, LiuXF, ZengJH, LiuXH, ChenYS, HeZY. Editing the MSTN gene of Guangdong Small Spotted pigs by using CRISPR-Cas9 system. Guangdong Agric Sci, 2017, 44( 02): 141- 148. |
| [14] | 王敏, 黄翔, 石翾, 刘小凤, 曾检华, 刘小红, 陈瑶生, 何祖勇. 应用CRISPR/Cas9编辑广东小耳花猪MSTN基因. 广东农业科学, 2017, 44(2): 141- 148. |
| [15] | ZhangZG, ZhangXL, LiJY. The excellent breeding of Chinese pigs and its contribution to the world pig industry. J Nat Resour, 1994, 9(1): 1- 8. |
| [15] | 张仲葛, 张晓岚, 李锦钰. 中国猪的优良种性及其对世界养猪业的贡献. 自然资源学报, 1994, 9(1): 1- 8. |
| [16] | RanFA, HsuPD, WrightJ, AgarwalaV, ScottDA, ZhangF. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013, 8(11): 2281- 2308. |
| [17] | LiuXF, LiuHB, WangM, LiRQ, ZengJH, MoDL, CongPQ, LiuXH, ChenYS, HeZY. Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs. Transgenic Res, 2019, 28(1): 141- 150. |
| [18] | QianLL, TangMX, YangJZ, WangQQ, CaiCB, JiangSW, LiHG, JiangK, GaoPF, MaDZ, ChenYX, AnXR, LiK, CuiWT. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Sci Rep, 2015,5:14435. |
| [19] | BerryC, ThomasM, LangleyB, SharmaM, KambadurR. Single cysteine to tyrosine transition inactivates the growth inhibitory function of Piedmontese myostatin. Am J Physiol Cell Physiol, 2002, 283(1): C135- 141. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
