CN117551729A - 一种通过mstn和fgf5双基因编辑抑制绵羊成肌细胞二次融合的方法与应用 - Google Patents
一种通过mstn和fgf5双基因编辑抑制绵羊成肌细胞二次融合的方法与应用 Download PDFInfo
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- CN117551729A CN117551729A CN202311462776.0A CN202311462776A CN117551729A CN 117551729 A CN117551729 A CN 117551729A CN 202311462776 A CN202311462776 A CN 202311462776A CN 117551729 A CN117551729 A CN 117551729A
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Abstract
本发明公开了一种通过MSTN和FGF5双基因编辑抑制绵羊成肌细胞二次融合的方法与应用。本发明提供了对MSTN和FGF5进行基因编辑的物质在如下任一种的应用:1)抑制成肌细胞的二次融合;2)降低肌管融合指数;3)降低单个肌管中的细胞核数;4)抑制骨骼肌卫星细胞中胞内Ca2+的释放;本发明提供了MSTN和FGF5双基因编辑绵羊骨骼肌卫星细胞,将其诱导分化,发现MSTN和FGF5双基因编辑抑制绵羊成肌细胞二次融合,MSTN和FGF5可以作为筛选抑制绵羊成肌细胞二次融合物质的作用靶点,在实践中用于提高动物产肉性能,为优质高产肉羊新品种的快速培育和肉质改良提供理论参考。
Description
技术领域
本发明属于生物技术领域,涉及一种通过MSTN和FGF5双基因编辑抑制绵羊成肌细胞二次融合的方法与应用。
背景技术
肌肉生长抑制素(Myostatin,MSTN)又被称为生长分化因子-8(Growthdifferentiation factor-8,GDF-8),是转化生长因子-β(Transforming growth factor-β,TGF-β)超家族的成员,其通过自分泌和旁分泌的方式负调控骨骼肌的生长发育。作为一种分泌蛋白,MSTN需要通过一系列的级联反应将其信号传递至细胞核。MSTN主要利用II型激活素A受体(Activin receptor kinase II-A,ActRIIA)和ActRIIB以及I型激活蛋白受体样激酶4(Activin receptor-like kinase 4,ALK4)和ALK5通过Smad2/3引起信号转导。除了经典的Smads信号转导途径外,MSTN也可依赖于丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)途径进行信号传递。例如,MSTN可通过TAK1-MKK6途径激活p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK),并抑制C2C12成肌细胞的增殖。MSTN也可通过结合其ActRIIB受体诱导下游JNK信号的激活,并进一步抑制C2C12成肌细胞的增殖与分化。此外,MSTN也可通过抑制Akt/TORC1/p70S6K信号传导抑制肌源性分化和肌管大小。
成纤维细胞生长因子5(Fibroblast growth factor 5,FGF5)属于成纤维细胞生长因子家族,是一种分泌型信号蛋白。FGF5被认为是毛囊发育过程中从生长期向退化期过渡的关键信号分子。目前,在猫、狗、仓鼠、驴和人类等几种物种中均发现了FGF5基因的自然突变,所有这些物种都显示出毛发长度的变化。与野生型绵羊相比,通过CRISPR Cas9生产的FGF5基因敲除绵羊的毛囊活性和羊毛长度显著增加。同样,在不影响其他性状的情况下,敲除FGF5基因导致克什米尔山羊次级毛囊数量和毛长度增加。此外,FGF5基因编辑的中国美利奴羊导致羊毛长度和产油量增加。总之,FGF5被认为是毛发生长的负调节因子,目前没有直接证据表明FGF5对肌肉表型的潜在调节作用。
长期以来,Ca2+一直被认为是哺乳动物肌肉融合的调节因子。内质网的短暂Ca2+耗竭与成肌细胞分化和融合有关,Ca2+敏感转录因子NFATc2可介导成肌细胞募集和肌管扩张。最近研究显示,胞内Ca2+水平升高对成肌细胞融合至关重要,新生肌管中的Ca2+信号先于肌管快速生长阶段,表明早期肌管中内质网释放的Ca2+可能促进成肌细胞二次融合和肌管扩张。然而,导致Ca2+介导的成肌细胞融合的信号级联仍不清楚。细胞内Ca2+水平通过各种Ca2+和电压门控通道调节,包括但不限于兰尼碱受体(Ryanodine receptors,RYR)。RYR是肌浆网的主要Ca2+释放通道,其调节Ca2+外流至细胞质。CaMKII是钙/钙调蛋白依赖性丝氨酸/苏氨酸激酶家族的成员,CaMKIIδ、CaMKIIγ和CaMKIIβ是骨骼肌中表达的主要亚型。
在哺乳动物中,骨骼肌卫星细胞经激活、增殖、分化为成肌细胞(myoblasts),成肌细胞再经终末分化、融合和肌核重排形成成熟肌管(myotubes),其中成肌细胞融合是一个两阶段过程。在第一阶段,成肌细胞与成肌细胞融合形成初级多核肌管。在第二阶段,额外的成肌细胞与已经形成的多核肌管细胞融合,这一过程通常被称为成肌细胞的二次融合。成肌细胞二次融合增加了单个肌管中的细胞核数,使肌纤维体积增加,但并不影响肌纤维数量,表现为肌纤维肥大。反之,抑制成肌细胞二次融合则有利于形成更多的初级多核肌管,使肌纤维数量增加并导致肌纤维增生表型,从而有利于培育肌纤维增生型高产肉性能绵羊。总之,成肌细胞的二次融合是骨骼肌卫星细胞成肌分化形成成熟肌管过程中重要的环节。因此,需要寻找调控该重要环节的靶点。
发明内容
本发明的目的是提供一种通过MSTN和FGF5双基因编辑抑制绵羊成肌细胞二次融合的方法与应用。
第一方面,本发明提供了以MSTN基因为基因编辑靶标的物质和以FGF5基因为基因编辑靶标的物质的应用在如下任一种的应用:
1)制备抑制成肌细胞二次融合的产品;
2)制备二次融合能力降低的细胞;在本发明的实施例中具体为来源于MSTN基因和FGF5基因编辑的动物的骨骼肌细胞,在本发明的实施例中MSTN基因和FGF5基因编辑的动物为MSTN基因和FGF5基因编辑均纯合(MSTN-/-和FGF5-/-)绵羊(命名为MF-/-绵羊)或MSTN基因和FGF5基因编辑杂合(MSTN+/-和FGF5+/-)绵羊(命名为MF+/-绵羊);
3)制备降低肌管融合指数的产品;
4)制备降低单个肌管中的细胞核数的产品;
5)制备抑制骨骼肌卫星细胞中胞内Ca2+的释放的产品;
6)制备抑制骨骼肌卫星细胞中CaMKIIα/δ的表达的产品;
7)制备抑制成肌细胞融合相关基因的表达的产品;在本发明的实施例中具体为MYMK、MYMX和/或Rac1;
8)培育高产肉性能动物(具体为肌纤维增生型高产肉性能动物)。
上文所述应用中,所述动物为哺乳动物,
进一步地,所述动物为绵羊。
第二方面,本发明提供了一种制备细胞模型的方法,包括如下步骤:利用基因编辑制备MSTN基因和FGF5基因双等位基因编辑的动物(MSTN-/-和FGF5-/-),再与野生型动物配种,生产子代,选取MSTN基因和FGF5基因编辑纯合(MSTN-/-和FGF5-/-)的动物或MSTN基因和FGF5基因编辑杂合(MSTN+/-和FGF5+/-)的动物,作为目标动物,分离获取所述目标动物的骨骼肌卫星细胞,即为细胞模型;
所述细胞模型的用途为筛选抑制成肌细胞二次融合的药物。
第三方面,本发明提供了第二方面所述方法制备的细胞模型在筛选药物中的应用;所述药物为抑制成肌细胞二次融合的药物。
第四方面,本发明提供了一种制备动物模型的方法,包括如下步骤:利用基因编辑制备MSTN基因和FGF5基因双等位基因编辑的动物(MSTN-/-和FGF5-/-),再与野生型动物配种,生产子代,选取MSTN基因和FGF5基因编辑纯合(MSTN-/-和FGF5-/-)的动物或MSTN基因和FGF5基因编辑杂合(MSTN+/-和FGF5+/-)的动物,作为目标动物,即为动物模型;所述动物模型的用途为筛选抑制成肌细胞二次融合的药物。
第五方面,本发明提供了第四方面所述方法制备的动物模型在筛选药物中的应用;所述药物为抑制成肌细胞二次融合的药物。
上文中,所述动物为哺乳动物,
进一步地,所述动物为绵羊。
本发明的有益效果在于:本发明提供了MSTN和FGF5双基因编辑绵羊骨骼肌卫星细胞,将其诱导分化,发现MSTN和FGF5双基因编辑抑制绵羊成肌细胞二次融合,MSTN和FGF5可以作为筛选抑制绵羊成肌细胞二次融合物质的作用靶点,在实践中用于构建筛选抑制绵羊成肌细胞二次融合物质的动物模型或用于提高动物产肉性能,为优质高产肉羊新品种的快速培育和肉质改良提供理论参考。
附图说明
图1为MSTN和FGF5双基因编辑抑制绵羊成肌细胞的鉴定结果。
图2为MSTN和FGF5双基因编辑抑制绵羊成肌细胞的二次融合。其中,A为WT和MF+/-绵羊肌管成肌分化标志因子MyoG和MyHC免疫荧光染色,标尺130μm;B为肌管融合指数;C为单个肌管中的细胞核数。
图3为MSTN和FGF5双基因编辑对钙依赖性转录信号通路和胞内Ca2+浓度的影响。其中,A为GM期WT和MF+/-细胞中CaMKIIα/δ蛋白表达水平。B为Western blot量化结果。
图4为MSTN和FGF5双基因编辑对胞内Ca2+浓度的影响。其中,A为GM期WT和MF+/-细胞胞内Ca2+信号分布;B为GM期WT和MF+/-细胞胞内Ca2+平均荧光强度。
图5为MSTN和FGF5双基因编辑对钙离子通道和成肌细胞融合因子的影响;其中,A为GM期Ca2+通道和成肌细胞融合相关基因mRNA表达水平;B为DM2期Ca2+通道和成肌细胞融合相关基因mRNA表达水平。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的MSTN和FGF5双基因编辑绵羊为将野生型绵羊中MSTN和FGF5双基因进行基因编辑,选取MSTN和FGF5基因与野生型绵羊中的MSTN(MSTN myostatin[Ovisaries(sheep)]Gene ID:443449,updated on 21-Jun-2023)和FGF5(FGF5 fibroblastgrowth factor 5[Ovis aries(sheep)]Gene ID:100642180,updated on 18-Aug-2023)相比均发生突变的基因编辑后的绵羊,即为MSTN和FGF5双基因编辑绵羊。
具体以如下MSTN和FGF5双基因编辑绵羊为例:按照专利ZL201910277213.1中记载制备:针对绵羊的MSTN基因设计了2个打靶位点Cr1和Cr2的sgRNA1(SEQ ID NO.1,)和sgRNA2(SEQ ID NO.2)。针对绵羊的FGF5基因设计了1个打靶位点Cr3的FGF5sgRNA(SEQ IDNO.3)。
扩增含有T7启动子的Cas9(基因序列如SEQ ID NO.4所示)及上述各个sgRNA序列,纯化后作为体外转录模板,得到转录后的sgRNA1、转录后的sgRNA2、转录后的sgRNA3和Cas9mRNA;再导入绵羊(杜泊绵羊)原核胚中,受精卵移植母体,生产后的子代羊分别进行MSTN和FGF5 PCR扩增,引物为MSTN基因突变PCR鉴定引物和FGF5基因突变PCR鉴定引物;测序扩增产物,选取MSTN和FGF5双等位基因突变的绵羊(与野生型绵羊中的MSTN和FGF5相比均发生突变),记作MSTN和FGF5双基因编辑绵羊(MSTN-/-和FGF5-/-)。
再以MSTN和FGF5双基因编辑公羊(MSTN-/-和FGF5-/-)作为父本,与野生型(MSTN+/+和FGF5+/+)母羊配种,生产MSTN和FGF5双基因编辑的杂合子F1代绵羊(基因型为MSTN+/-和FGF5+/-,又称为MF+/-绵羊)。
实施例1、MSTN和FGF5双基因编辑抑制绵羊成肌细胞的二次融合
一、MF+/-(MSTN+/-和FGF5+/-)绵羊骨骼肌卫星细胞的制备
1、绵羊骨骼肌卫星细胞的制备
(1)从配种后的母羊中剖腹产取出3月龄左右绵羊胎儿,取胎儿后肢肌肉组织,保存于含10%双抗的DMEM/F-12培养基,低温运回实验室。
(2)取若干小块肌肉组织,依次于酒精、PBS中浸泡30s,完全剔除脂肪、筋膜和肌膜等结缔组织。
(3)眼科剪尽量剪碎组织块,用1mL移液器转移至50mL离心管,加预冷PBS静置5min,1000rpm离心5min,弃上清。
(4)按1g组织需2-3mL酶的比例,加入0.2% II型胶原酶,于37℃消化1h,每10min振荡1次,消化至混浊状。若过于粘稠,加PBS稀释。1000rpm离心10min,小心吸弃上清。
(5)按1g组织需2-3mL酶的比例,加入0.25%胰酶,于37℃消化30min,每10min振荡1次。
(6)加入等体积增殖培养基(DMEM/F-12+20% FBS+1%双抗)终止消化,颠倒混匀,依次滤过纱布、100目、200目和400目细胞筛。
(7)滤液于1000rpm离心10min,弃上清。细胞沉淀用增殖培养基重悬于50mL离心管,于37℃5% CO2培养箱培养2-3h,每30min颠倒混匀1次。
(8)将细胞接种于培养皿,37℃5% CO2培养箱差速贴壁培养不超过30min,未贴壁细胞为绵羊骨骼肌卫星细胞,反复差速贴壁2-3次,得到骨骼肌卫星细胞。
2、MF+/-绵羊骨骼肌卫星细胞的鉴定
将上述获得的骨骼肌卫星细胞进行MSTN和FGF5基因型鉴定,具体如下:
提取骨骼肌卫星细胞基因组DNA,用如下鉴定引物分别进行PCR扩增:
MSTN基因突变PCR鉴定引物序列为:MSTN-F:AAGTCAAGGTAACAGACACACC,MSTN-R:TGTGTTTTAGGAAGCTATGAAC。
FGF5基因突变PCR鉴定引物序列为:FGF5-F:TGCAAGTTCAGGGAGCGATT,FGF5-R:ATCCCTGTATGCACCAAGCA。
将上述PCR扩增产物亚克隆送去测序,结果如图1所示,A为MSTN基因基因编辑靶点和测序峰图,B为MSTN基因编辑后突变形式(Δ3表示MSTN基因缺失3个碱基,3/10表示10个克隆中的3个;WT为野生型MSTN基因),C为FGF5基因基因编辑靶点和测序峰图,D为FGF5基因编辑后突变形式(Δ5表示FGF5基因缺失5个碱基,5/10表示10个克隆中的5个;WT为野生型FGF5基因)。选取符合如下特征的细胞,命名为MF+/-绵羊骨骼肌卫星细胞,该细胞来源的胎儿即为MF+/-绵羊:MSTN基因突变PCR鉴定引物得到的扩增产物与野生型MSTN基因相比,发生杂合突变(如图1中的B所示,基因型为MSTN+/-),且FGF5基因突变PCR鉴定引物得到的扩增产物与野生型FGF5基因相比,发生杂合突变(如图1中的D所示,基因型为FGF5+/-)。
二、MSTN和FGF5双基因编辑抑制绵羊成肌细胞的二次融合
培养WT绵羊骨骼肌卫星细胞和MF+/-绵羊骨骼肌卫星细胞至汇合度达80%左右,更换培养基为分化培养基,连续培养6天,进行成肌诱导分化,具体方法如下:
1、绵羊骨骼肌卫星细胞培养与成肌诱导分化
(1)于液氮中取出冻存的绵羊骨骼肌卫星细胞(记作野生型绵羊骨骼肌卫星细胞,其为分离获得,分离方法参考文献:Doi:10.1089/dna.2022.0574)和上述一制备的MF+/-绵羊骨骼肌卫星细胞,迅速置于37℃水浴中快速摇晃1-2min。至冻存液融化后加入等体积增殖培养基(DMEM/F-12+20% FBS+1%双抗),重悬混匀,2000rpm离心5min收集细胞沉淀。
(2)弃培养基,细胞沉淀用增殖培养基重悬后按30%密度接种于培养皿,“十”字晃匀,置37℃5% CO2培养箱培养。
(3)细胞汇合至80-90%左右,弃培养基,PBS清洗1次,加适量0.25%胰酶,置37℃5% CO2培养箱消化3min左右。
(4)观察到大部分细胞脱落时,立即加入等体积增殖培养基终止消化,转移细胞悬液至离心管,2000rpm离心5min。
(5)弃培养基,细胞沉淀用增殖培养基重悬,按30%密度接种于新的培养皿或细胞培养板,37℃5% CO2培养箱培养(记作GM期细胞)。
(6)细胞汇合度达70%左右,更换培养基为成肌分化诱导培养基(DMEM+2%HS+1%双抗)诱导细胞成肌分化,收集不同成肌诱导分化时间(从更换成肌分化诱导培养基起记作诱导分化第0天,记作DM0)的贴壁细胞样品。
绵羊骨骼肌卫星细胞成肌诱导分化第2天的细胞记作DM2期肌管细胞。
绵羊骨骼肌卫星细胞成肌诱导分化第6天的细胞记作DM6期肌管细胞。
增殖培养基配方如下:DMEM/F-12、体积百分含量20% FBS和体积百分含量1%双抗(10000U/mL;Gbico,货号15140122)。
成肌分化诱导培养基(DMEM+2% HS+1%双抗)配方如下:DMEM、体积百分含量2%HS(Gbico,货号:26050088)+体积百分含量1%双抗。
2、采用免疫荧光染色技术对诱导分化第6天细胞的成肌分化标志因子MyoG和MyHC进行双重免疫荧光染色
免疫荧光染色每组试验至少设置3个生物学重复,以48孔板为例,具体操作步骤如下:
(1)将上述1诱导分化第6天细胞弃培养基,PBS清洗细胞1-2次,每孔加入100μL4%多聚甲醛,室温固定细胞30min。PBS清洗细胞3次,每次5min。
(2)每孔加入100μL含0.1% Triton X-100的PBS,室温通透细胞20min(由于Triton X-100会破坏细胞膜,因此细胞膜上表达的抗原省略此步骤)。PBS清洗细胞3次,每次5min。
(3)每孔加入100μL 5% Normal Goat Serum,室温封闭30min,弃封闭液,不洗。按抗体说明书稀释一抗,每孔加入100μL稀释后的一抗,4℃过夜孵育。
(4)PBS清洗细胞3次,每次5min。每孔加入100μL相应的荧光二抗,37℃避光孵育1h。PBS清洗细胞3次,每次5min。
(5)每孔加入100μL即用型DAPI溶液,室温避光孵育5min,PBS清洗细胞1-2次。
(6)每孔加入100μL PBS,采用Echo Revolve荧光显微镜成像存图,每孔至少取5个不同视野。
结果显示,与WT细胞相比,DM6期MF+/-组细胞的肌管融合指数(图2A和图2B)和单个肌管中的细胞核数(图2A和图2C)均极显著(P<0.001)降低,表明MSTN和FGF5双基因编辑抑制绵羊成肌细胞的二次融合。
实施例2、MSTN和FGF5双基因编辑对CaMKII的影响
提取GM期MF+/-绵羊骨骼肌卫星细胞和GM期WT绵羊骨骼肌卫星细胞的总蛋白,通过Western blot检测骨骼肌卫星细胞细胞钙-钙调蛋白依赖性蛋白激酶II(Calcium-calmodulin dependent protein kinase II,CaMKII)的表达情况。
具体如下:
(1)总蛋白提取:新鲜组织样品20mg,液氮研磨,转移至离心柱,加入200μL含蛋白酶抑制剂混合物的裂解液,研磨30-60次,室温孵育2min,14000×g离心30s收集裂解物。细胞样品用预冷PBS清洗,每个6孔板加入200μL含蛋白酶抑制剂混合物的裂解液裂解细胞,转移裂解物至离心柱,14000×g离心30s收集裂解物。
(2)蛋白浓度测定:采用BCA蛋白定量试剂盒进行蛋白浓度测定。
(3)蛋白变性:用6×蛋白上样缓冲液稀释蛋白,调整上样量相同,100℃变性10min。
(4)SDS-PAGE电泳:按PAGE凝胶快速制备试剂盒说明书制胶,按试验需求上样,于80V恒压电泳30-40min,至溴酚蓝到达分离胶后,120V恒压电泳90min左右,至溴酚蓝到达胶的底端。
(5)电泳结束,去除浓缩胶,以320mA恒定电流转膜1.5h。
(6)转膜结束,用5%脱脂奶粉封闭1h,TBST清洗膜1次,每次5min。
(7)按抗体稀释比例稀释一抗(CaMKII alpha/delta Antibody,AffinityBiosciences,目录号AF6493;Mouse Anti-GAPDH mAb,北京中杉金桥生物技术有限公司,目录号TA-08),于4℃将膜与一抗孵育过夜。
(8)TBST清洗膜3次,每次5min。室温下于已稀释的二抗(辣根酶标记山羊抗小鼠IgG,北京中杉金桥生物技术有限公司,目录号ZB-2305;辣根酶标记山羊抗兔IgG,北京中杉金桥生物技术有限公司,目录号ZB-2301)中孵育膜1h,TBST清洗膜3次,每次5min。
(9)向PVDF膜滴加ECL超敏发光液,采用蛋白化学发光系统成像存图。
结果如图3所示,与WT绵羊骨骼肌卫星细胞相比,GM期MF+/-绵羊骨骼肌卫星细胞中CaMKIIα/δ蛋白表达水平显著(P<0.05)降低(图3A-B),表明MSTN和FGF5双基因编辑抑制绵羊骨骼肌卫星细胞中CaMKIIα/δ的表达。
实施例3、MSTN和FGF5双基因编辑对胞内Ca2+浓度的影响
使用5μM的Fluo-3 AM孵育WT和MF+/-绵羊骨骼肌卫星细胞,采用流式细胞仪检测Fluo-3 AM的荧光信号。
具体如下:
(1)GM期绵羊骨骼肌卫星细胞接种于6孔板,增殖培养基培养至细胞数达1×106,0.25%胰酶消化,PBS清洗1次。
(2)细胞沉淀中加入500μL终浓度为5μM的Fluo-3 AM,重悬细胞。37℃避光孵育30min,PBS清洗1次。
(3)加入500μL DMEM重悬细胞沉淀,流式细胞仪检测。
结果如图4所示,与WT绵羊骨骼肌卫星细胞相比,GM期MF+/-绵羊骨骼肌卫星细胞内Ca2+浓度显著(P<0.05)降低,表明MSTN和FGF5双基因编辑抑制绵羊中胞内Ca2+的释放。
实施例4、MSTN和FGF5双基因编辑对钙离子通道与成肌细胞融合的影响
高通量Ca2+通道RYR负责将Ca2+从内质网快速大量释放到胞浆中。
提取GM期和DM2期的MF+/-绵羊骨骼肌卫星细胞和WT绵羊骨骼肌卫星细胞的总RNA提取,反转录合成cDNA,进行荧光定量PCR检测。具体如下:
1、绵羊骨骼肌细胞总RNA提取
(1)贴壁细胞样品,吸弃培养基,预冷PBS清洗细胞1-2次,加入1mL TRIzol,吹打混匀并转移至1.5mL无酶离心管。
(2)室温放置5-10min,加入0.2mL氯仿,涡旋15s,室温静置3min。12000rpm 4℃离心10min。
(3)转移上层水相至无酶离心管,加入等体积异丙醇,充分混匀,室温静置20min。
(4)12000rpm 4℃离心5min,弃上清。加入1mL 75%乙醇洗涤沉淀。12000rpm 4℃离心3min,弃上清,室温干燥5-10min。
(5)加入30-50μL RNase Free dH2O,充分溶解RNA,-80℃保存。
2、cDNA第一链合成与实时荧光定量PCR(RT-qPCR)
(1)利用PrimeScriptTMRTreagent KitwithgDNAEraser进行cDNA第一链的合成,具体步骤如下:
A.按表1所示于冰上配制去除基因组DNA反应体系,于42℃反应2min,冰浴2-3min。
表1为去除基因组DNA反应体系
B.按表2所示于冰上配制反转录反应体系,分别加至上述去除基因组DNA的反应体系,于37℃反应15min,85℃反应5s。反应结束后,将cDNA第一链产物稀释5倍,保存于-20℃。
表2为反转录反应体系
(2)RT-qPCR
A.按表3所示配制RT-qPCR反应体系,每个样品设置4个复孔。
表3为RT-qPCR反应体系
B.RT-qPCR反应程序如表4所示。
表4为RT-qPCR反应程序
C.FOSL1和内参基因GAPDH的RT-qPCR引物如表5所示。
表5为RT-qPCR引物
结果如下:与WT细胞相比,MF+/-绵羊骨骼肌卫星细胞中RYR1 mRNA表达水平显著(P<0.05)降低(图5A),RYR3 mRNA表达水平在MF+/-绵羊骨骼肌卫星细胞和肌管细胞中均极显著(P<0.001)降低(图5B)。鉴于胞内Ca2+浓度与成肌细胞融合相关,本发明也检测了成肌细胞融合相关基因MYMK、MYMX和Rac1的表达水平。结果显示,与WT细胞相比,MYMK和MYMX mRNA表达水平在MF+/-绵羊骨骼肌卫星细胞和肌管细胞中均极显著(P<0.01)降低(图5A-B)。
表明MSTN和FGF5双基因编辑通过抑制胞内Ca2+的释放,从而抑制成肌细胞的二次融合。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.以MSTN基因为基因编辑靶标的物质和以FGF5基因为基因编辑靶标的物质的应用在如下任一种的应用:
1)制备抑制成肌细胞二次融合的产品;
2)制备二次融合能力降低的细胞;
3)制备降低肌管融合指数的产品;
4)制备降低单个肌管中的细胞核数的产品;
5)制备抑制骨骼肌卫星细胞中胞内Ca2+的释放的产品;
6)制备抑制骨骼肌卫星细胞中CaMKIIα/δ的表达的产品;
7)制备抑制成肌细胞融合相关基因的表达的产品;
8)培育高产肉性能动物。
2.一种制备细胞模型的方法,包括如下步骤:利用基因编辑制备MSTN基因和FGF5基因双等位基因编辑的动物,再与野生型动物配种,生产子代,选取MSTN基因和FGF5基因编辑纯合的动物或MSTN基因和FGF5基因编辑杂合的动物,作为目标动物,分离获取所述目标动物的骨骼肌卫星细胞,即为细胞模型;
所述细胞模型的用途为筛选抑制成肌细胞二次融合的药物。
3.权利要求2所述方法制备的细胞模型在筛选药物中的应用;所述药物为抑制成肌细胞二次融合的药物。
4.一种制备动物模型的方法,包括如下步骤:利用基因编辑制备MSTN基因和FGF5基因双等位基因编辑的动物,再与野生型动物配种,生产子代,选取MSTN基因和FGF5基因编辑纯合的动物或MSTN基因和FGF5基因编辑杂合的动物,作为目标动物,即为动物模型;所述动物模型的用途为筛选抑制成肌细胞二次融合的药物。
5.权利要求4所述方法制备的动物模型在筛选药物中的应用;所述药物为抑制成肌细胞二次融合的药物。
6.根据权利要求1或5所述的应用或权利要求2或4所述方法,其特征在于:
所述动物为哺乳动物,
进一步地,所述动物为绵羊。
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