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通讯作者:

曹新,E⁃mail:caoxin@njmu.edu.cn

中图分类号:R784

文献标识码:A

文章编号:1007-4368(2021)01-004-07

DOI:10.7655/NYDXBNS20210102

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参考文献 13
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参考文献 14
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参考文献 15
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参考文献 16
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参考文献 17
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参考文献 20
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目录contents

    摘要

    目的:采用CRISPR/Cas9基因编辑技术构建G蛋白偶联受体相关分选蛋白2(G protein⁃coupled receptor associated sorting protein 2,GPRASP2)基因敲除的猪胎儿成纤维细胞(porcine fetal fibroblast,PFF),为构建GPRASP2基因敲除巴马小型猪模型提供相应的供体细胞。方法:采用生物信息学方法对人与猪 GPRASP2 基因进行亲缘性和同源性分析,预测人与猪 GPRASP2基因编码的氨基酸序列和蛋白二级结构;设计合成靶向巴马猪GPRASP2基因编码区上游和下游的单导向RNA(sin⁃ gle guide RNA,sgRNA),以pX330质粒为载体,构建含有Cas9骨架的重组打靶质粒,并将此重组质粒转染至PFF中,G418药物筛选阳性单克隆细胞,测序分析其基因型。结果:生物信息学分析提示人/猪GPRASP2亲缘关系相近,同源性较高,且主要功能结构域Arm2的二维和三维结构相似。构建了靶向猪GPRASP2基因的重组打靶质粒并转染PFF,通过药物筛选、基因型分析和Western blot验证,成功获得GPRASP2基因敲除单克隆PFF。结论:人/猪GPRASP2基因亲缘关系相近且高度同源;采用 CRISPR/Cas9介导的基因编辑技术成功构建GPRASP2基因敲除的单克隆PFF,为建立GPRASP2基因敲除巴马小型猪模型奠定了前期基础。

    Abstract

    Objective:To construct G protein ⁃ coupled receptor associated sorting protein 2(GPRASP2)⁃ modified procine fetal fibroblasts(PFFs)as donor cells for the generation of GPRASP2 ⁃ disrupted Bama miniature(BM)pigs. Methods:Bioinformatics methods were applied to phylogenetic and homologue analysis of human/porcine GPRASP2,and the secondary structures of human/ porcine GPRASP2 proteins were predicted. Two single⁃guide RNAs(sgRNAs),targeting the upstream/downstream of coding sequence of the porcine GPRASP2,were designed,synthesized and ligated to pX330 plasmid. The recombinant plasmids containing Cas9 backbone were transfected into PFFs. Viable cell colonies were obtained using G418 screening and subjected to genotyping via direct PCR ⁃ based sequencing. Results:The human and porcine GPRASP2 proteins are evolutionarily closer,highly homologous,and predicted to have the similar functional Arm2 domains via 2D and 3D structure modeling. CRISPR/Cas9⁃ sgRNA expression vectors targeting porcine GPRASP2 were constructed and transfected into PFFs. GPRASP2⁃deficient monoclonal PFFs were obtained by drug screening,genotypic analysis and Western blot assay. Conclusion:The human/porcine GPRASP2 proteins are evolutionarily closer and highly homologous. The GPRASP2⁃deficient cells were successfully constructed via CRISPR/Cas9 mediated gene editing,which provided a substantial foundation for the generation of GPRASP2⁃disrupted BM pigs.

  • G蛋白偶联受体(G protein ⁃ coupled receptor, GPCR)是人类基因组中最庞大的膜蛋白家族,它们是一类具有7次跨膜螺旋结构的超家族[1],主要通过与配体结合并发生内吞后发挥作用,可调控生物体内许多重要的生理过程。G蛋白偶联受体相关分选蛋白(GPCR associated sorting protein,GASP)主要在GPCR内吞后的分选过程发挥重要作用,通过与受体的羧基端相互结合,从而介导GPCR进入降解或再循环途径,从而调控相关信号转导[2]。GPRASP2为GASP家族成员(即GASP2),参与GPCR活性调节,并与肿瘤发生、细胞生长及衰老、生理调节和溶酶体降解等生理过程相关[2]。本课题组在1个X连锁隐性遗传耳聋家系中首次定位和发现GPRASP2基因突变(c.1717_1718GC>AA,p.A573N)与综合征性耳聋(syndromic hearing loss,SHL)的发生相关[3]。为进一步推进对GPRASP2基因突变/功能缺陷致聋的分子病因学的理解,还需要在模型动物水平进行耳聋基因型/表型相关性的研究和GPRASP2功能的探讨。

  • 随着基因编辑技术的发展,构建基因缺陷的动物模型为从病理与病因学上研究遗传性耳聋提供了有效途径。小鼠等啮齿类模型动物的内耳发育、遗传和解剖结构与人类差别较大[4],无法精确模拟人内耳的生理功能和病理过程。猪在分子进化上与人亲缘关系相近,在遗传、生理生化、器官发育和病程发展等方面,特别在内耳发育、听器结构和功能上与人相似[5-6]:猪出生时耳蜗形态已基本发育成熟并已具备正常听力,这与人胚胎时期的内耳发育过程一致[7-8];猪的内耳耳蜗形态以及螺旋器结构和功能与人相似;猪的听阈和敏感听力范围与人接近[9],具有相似的听性脑干反应(auditory brainstem response,ABR)[10];巴马小型猪体型大小合适,遗传背景明晰,应用于遗传性耳聋的研究更具优势[10]。本研究采用CRISPR/Cas9介导的基因编辑技术成功获得猪GPRASP2基因敲除的猪胎儿成纤维细胞(porcine fetal fibroblasts,PFF),为构建GPRASP2基因缺陷的巴马小型猪模型和在体水平探索GPRASP2的致聋机制奠定了实验基础。

  • 1 材料和方法

  • 1.1 材料

  • PFF由南京医科大学江苏省异种移植重点实验室提供;pX330质粒(Addgene423230)、带G418抗性标记的pCMV td ⁃tomato质粒(Clontech公司,日本);BbsⅠ限制性内切酶和T7EN1酶(New England Biolabs公司,美国);琼脂糖凝胶回收试剂盒(Qiagen公司,德国);质粒小提中量试剂盒和DH5α感受态细胞(北京天根生化科技公司);Basic NucleofectorTM Kits和细胞转染仪(Lonza公司,德国);DMEM培养基、胎牛血清、胰酶、青/链霉素双抗和PBS缓冲液 (Gibco公司,美国);pMD18⁃T载体(TaKaRa公司,日本);引物和磷酸化的寡核苷酸序列由南京金斯瑞公司合成。

  • 1.2 方法

  • 1.2.1 人/猪GPRASP2的生物信息学分析

  • 采用MEGA7.0软件,选取包括猪和人等20种物种进行GPRASP2基因亲缘性分析,并绘制GPRASP2的系统进化树。采用GeneDoc软件,比对人/猪GPRASP2氨基酸序列,并进行同源性和保守性分析。

  • 采用DNAstar软件中Protean模块对人/猪GPRASP2蛋白进行二级结构分析,以Chou⁃Fasman算法预测蛋白α螺旋、β折叠、β转角的比例。采用Swiss⁃Model在线工具对人/猪GPRASP2蛋白的三维结构进行模拟(https://www.swissmodel.expasy.org/),因Protein Data Bank(PDB)数据库中未检索到GPRASP2同源蛋白的结构,故采用从头计算法对人/猪GPRASP2蛋白主要功能结构域Arm2(含250个氨基酸残基)进行三维结构模拟。

  • 1.2.2 CRISPR/Cas9的靶点设计和打靶载体构建

  • 利用CRISPR靶点在线设计工具(http://crispr.mit.edu/),分别在猪GPRASP2基因编码区(coding sequence,CDS)的上游和下游各设计1对20bp左右的sgRNA寡核苷酸序列(sgRNA oligo,表1),由南京金斯瑞合成5′端磷酸化修饰的sgRNA oligo。

  • 表1 GPRASP2靶点位置及sgRNA寡核苷酸序列

  • Table1 GPRASP2targeting loci and sgRNA oligonucleo⁃ tide sequence

  • 载体构建步骤主要包括oligo退火、载体酶切和连接[11-13]。将合成的sgRNA oligo以去离子水稀释至100 μmol/L,反应体系(10 μL):正链oligo 1 μL,反链oligo 1 μL,去离子水8 μL;PCR仪设置退火程序:37℃孵育30min,95℃孵育5min,再以5℃/min速度逐渐降温至25℃;退火后将oligo稀释250倍。用BbsⅠ限制性内切酶对pX330质粒进行线性化,并连接退火产物,再转化感受态DH5α,挑单克隆菌落至摇管,37℃培养12~16h,分装1mL的菌液送测序。测序验证后冻存菌液,并小提质粒。

  • 1.2.3 细胞转染和单克隆细胞的筛选

  • PFF培养于含16%胎牛血清的DMEM培养基含中,待细胞汇合度达到90%后,以0.05%胰蛋白酶消化并离心收集细胞;取1 μg pCMV td⁃tomato分别与空载pX330质粒、靶向猪GPRASP2基因CDS上游和下游的重组pX330⁃CDS⁃UP/pX330⁃CDS⁃DOWN质粒各5 μg共转染PFF;参照Lonza核转试剂盒说明书配制核转液,使用程序U⁃023进行核转,转染后24h加1mg/mL的G418进行药筛培养,隔天换液,并根据细胞生长状态调整G418用药浓度。9~10d后,显微镜观察单克隆位置,并在皿底标记;弃皿中培养液,PBS清洗2遍,将克隆环放置于皿底单克隆位置,加0.05%胰蛋白酶消化细胞,转接于24孔板中,待细胞长满后再转接至12孔板中培养,孔板盖上标记克隆编号和日期;12孔板中细胞长满后,取部分细胞用NP40裂解液裂解,提取基因组DNA,PCR扩增靶点区域,CDS1上游引物5′ ⁃ TTCTGCACTCTGTTG⁃ GCTGAG ⁃ 3′,下游引物5′ ⁃ AGCAGCAGAAC⁃ CAGACTCATT ⁃3′,扩增产物长度722bp。反应条件:95℃预变性3min;95℃ 15s,60℃ 15s,72℃ 1min,35个循环;PCR产物经切胶回收,连接pMD18⁃ T载体,连接产物转化感受态DH5α中,每板挑取12个菌落送公司测序;测序结果与猪GPRASP2基因序列(XM⁃003135261.4)进行比对,鉴定得每个克隆细胞的基因型。

  • 1.2.4 重组载体打靶效率验证

  • 提取转染细胞的基因组DNA,同前PCR扩增;切胶回收PCR产物(5 μL),加入2 μL 10× NEBuffer 2.0,用Nuclease⁃free水补足至19 μL,在PCR仪中进行退火反应:95℃ 10min,再逐渐冷却至室温; 退火结束后,取9.5 μL退火产物加入0.5 μL T7EN1酶,剩余9.5 μL退火产物加0.5 μL去离子水(即不加酶的对照组),混匀后37℃下反应15min;加入0.5 μL蛋白酶K使T7EN1酶失活,采用1.5%琼脂糖凝胶电泳分析和检测打靶效率。根据条带灰度值,计算细胞转染敲除效率,敲除效率=100×[1-(1-裂解产物占比)1/2 ]。

  • 1.2.5 Western blot验证

  • 分别取野生型和GPRASP2基因敲除的PFF,加入含有蛋白酶抑制剂的RIPA裂解液后超声破碎和低温离心,提取总蛋白,经SDS⁃PAGE胶分离,转膜至PVDF膜上,用5%脱脂奶粉室温封闭2h,加GPRASP2抗体(1∶1 000,Abcam公司,英国)和β⁃tu⁃ bulin抗体(1∶1 000,北京翼飞雪),4℃孵育过夜, TBST洗膜后室温孵育二抗(羊抗兔或羊抗鼠)2h, TBST洗膜后,化学发光仪(Bio⁃Rad公司,美国)检测蛋白表达情况。

  • 2 结果

  • 2.1 人/猪GPRASP2基因的分子进化关系与同源性分析

  • 包含猪和人在内20个物种的GPRASP2系统进化树如图1A所示,分值越高的节点其置信度也越高。进化树显示人/猪GPRASP2在分子进化上亲缘关系更近,且人/猪GPRASP2蛋白同源性较高,氨基酸序列一致性达86%(图1B),且主要功能结构域Arm2的氨基酸序列一致性高达94%。

  • 2.2 人/猪GPRASP2蛋白结构分析

  • 采用DNAstar软件中Protean模块的Chou⁃Fas⁃ man算法对人/猪GPRASP2蛋白进行二级结构分析。人/猪GPRASP2蛋白及其主要功能结构域Arm2的二级结构具有相近的α螺旋、β折叠、β转角的数量(表2),提示人/猪GPRASP2蛋白的二级结构相似。蛋白三维结构模拟显示人/猪GPRASP2的主要功能结构域Arm2的三维结构相似。考虑到人/猪GPRASP2在分子进化上亲缘关系相近且高度同源,推测人/猪GPRASP2蛋白具有相似的生物学功能,这还需从在体水平进一步验证。

  • 2.3 重组打靶质粒和GPRASP2基因敲除PFF的基因型鉴定

  • 测序结果如图2所示,pX330中分别插入了GPRASP2基因CDS上游和下游靶点的sgRNA序列。

  • 经测序鉴定,共获得42个阳性单克隆,其中5个为双等位突变的阳性单克隆(表3),包含5种等位基因突变型(图3),4种导致GPRASP2编码氨基酸的移码和蛋白截短。

  • 2.4 GPRASP2基因敲除效率的验证

  • 如图4所示,转染pX330⁃CDS⁃UP和pX330⁃CDS ⁃DOWN重组打靶质粒的PFF对应的PCR产物均可观察到明显剪切条带,敲除效率分别为20%和15%,选取敲除效率较高的pX330⁃CDS⁃UP重组质粒用于后续转染和单克隆PFF筛选。

  • 2.5 GPRASP2基因敲除PFF的Western blot验证

  • 采用Western blot随机验证其中1株GPRASP2基因敲除PFF细胞系(GPRASP2c.113_114delCT/c.113_114delCT)中GPRASP2蛋白的表达情况。如图5所示,在GPRASP2基因敲除PFF细胞中未检测到GPRASP2蛋白表达,表明GPRASP2基因敲除PFF构建成功,可用于后续的体细胞核移植。

  • 图1 GPRASP2系统进化树和人/猪GPRASP2蛋白氨基酸同源性分析

  • Fig.1 GPRASP2phylogenetic tree and homology analysis of human and porcine GPRASP2

  • 表2 GPRASP2蛋白及其Arm2结构域的二级结构分析

  • Table2 Analysis of the secondary structures of GPRASP2protein and Arm2domain

  • 图2 重组打靶质粒测序验证图

  • Fig.2 Sequencing of recombinant vectors

  • 3 讨论

  • 本课题组在中国1个X⁃连锁遗传性SHL大家系中成功定位和克隆了1个新的致聋基因GPRASP2,该家系患者均为男性且携带GPRASP2突变,表现为重度以上听力损失伴外耳廓畸形、内耳道狭窄/闭锁以及眼睑下垂等表型,并在家系成员中呈现典型的遗传共分离[3]。GPRASP2基因定位于人染色体Xq22.1,编码区位于第5外显子,共编码838个氨基酸,其羧基端含有高度保守的Arm2功能结构域,该结构域主要通过与多个GPCR的羧基末端结构域相互作用,从而特异性靶向GPCR进入溶酶体降解或再循环途径[14]。本研究提示人/猪GPRASP2在分子进化上亲缘关系相近并高度同源,且人/猪GPRASP2蛋白及其主要功能结构域Arm2在二级结构和三级结构上高度相似,推测GPRASP2基因在人/猪相应组织器官(包括内耳)中可能具有相似的生物学功能。

  • 图3 GPRASP2基因敲除PFF的等位基因型分析

  • Fig.3 The allelic genotypes of GPRASP2⁃knockout PFF

  • 表3 GPRASP2基因敲除PFF单克隆细胞基因型

  • Table3 Genotypes of GPRASP2⁃knockout monoclonal PFF

  • 图4 猪GPRASP2基因敲除靶点的T7EN1酶切验证

  • Fig.4 T7EN1cleavage assay of target sites in porcine GPRASP2

  • 图5 GPRASP2基因敲除PFF的Western blot验证

  • Fig.5 Western blot assay of GPRASP2⁃deficient PFF

  • GPRASP2作为GASP家族成员(GASP2)在GPCR内吞后的分选过程中发挥着重要作用。GP⁃ CR经配体刺激后,质膜表面的蛋白受体经G蛋白偶联受体激酶磷酸化以及β⁃arrestins作用下与G蛋白解偶联,通过网格蛋白有被小泡的协助完成内吞,内吞后的分选途径一般包括再循环和降解[15]。目前已经鉴定出多个受体再循环途径以及GPCR内吞后进入溶酶体降解途径的GASP[15⁃16],涉及多方面的生理功能。有研究表明GASP家族蛋白可作为肿瘤的分子标志物指示肿瘤的发生发展,GASP1在多种类型的肿瘤患者血清中都具有较高的表达量,包括乳腺癌、脑瘤、肝癌以及肺癌等[17];GASP2和GASP3在术前和术后头颈鳞状细胞癌患者中的表达具有显著差异[18];Horn等[19] 研究发现GPRASP2羧基端部分与亨廷顿蛋白(htt)的氨基端(polyQ)相互作用,形成htt⁃GPRASP2复合物,参与受体胞吞作用和突触后信号转导,影响受体的转运,而这一过程与亨廷顿舞蹈症发生有较大相关性;Edfawy等[20] 研究发现GPRASP2能够通过代谢型谷氨酸受体(mGluR) 信号通路调控神经系统发育,且敲除GPRASP2基因导致小鼠出现自闭症谱系障碍样行为[20]。现有研究提示GPRASP2与神经系统疾病相关;本课题组则在人类家系中首次关联了GPRASP2基因突变/功能缺陷与SHL的发生相关[3]

  • 为精确模拟GPRASP2突变导致的SHL表型,从在体水平研究GPRASP2的功能及其突变致聋的机制,本研究拟选择巴马小型猪构建耳聋动物模型。猪是除了灵长类动物以外在进化关系上与人最接近的实验动物,并在诸多器官(包括听觉器官)的解剖结构和发育遗传方面高度相似[13]。采用基因编辑技术构建基因缺陷的耳聋模型猪可精确模拟人遗传性耳聋的疾病表型和病理过程,如杨仕明课题组利用乙基亚硝基脲(ENU)诱变构建了MITF基因敲除小型猪模型,成功复制了人Mondini畸形病伴听力损失的临床表型[21];本课题组基于CRIS⁃ PR/Cas9介导的基因编辑技术成功构建了OSBPL2基因敲除的巴马小型猪模型,首次精确模拟了人OSBPL2基因突变导致的渐进性听力损失的临床表型(DFNA67)和内耳听毛细胞的病理过程[13]

  • 本研究利用CRISPR/Cas9技术成功构建了GPRASP2基因敲除的PFF细胞系,为后续体细胞核移植和重组胚胎构建提供合适的供体细胞,为构建GPRASP2基因敲除的巴马小型猪模型奠定了前期基础,以期为进一步探索GPRASP2基因突变致聋机制与基因治疗提供合适的大动物模型。

  • 参考文献

    • [1] MYKYTYN K,ASKWITH C.G⁃protein⁃coupled receptor signaling in cilia[J].Cold Spring Harb Perspect Biol,2017,9(9):a028183

    • [2] ABU⁃HELO A,SIMONIN F.Identification and biological significance of G protein⁃coupled receptor associated sort⁃ ing proteins(GASPs)[J].Pharmacol Ther,2010,126(3):244-250

    • [3] XING G,YAO J,LIU C,et al.GPRASP2,a novel caus⁃ ative gene mutated in an X ⁃ linked recessive syndromic hearing loss[J].J Med Genet,2017,54:426-430

    • [4] STEPHANIE E,DIETMAR H,KATHARINA S,et al.Co⁃ chlea ⁃ specific deletion of Cav1.3 calcium channels ar⁃ rests inner hair cell differentiation and unravels pitfalls of conditional mouse models[J].Front Cell Neurosci,2019,13:225

    • [5] GUO W,YI H,REN L,et al.The morphology and electro⁃ physiology of the cochlea of the miniature pig[J].Anat Rec(Hoboken),2015,298:494-500

    • [6] 陈伟,陈磊,杨仕明,等.荣昌猪遗传性听力缺陷家系的发掘与应用[J].中华耳科学杂志,2016,14(1):10-14

    • [7] HOFFSTETTER M,LUGAUER F,KUNDU S,et al.Mid⁃ dle ear of human and pig:a comparison of structures and mechanics[J].Biomed Tech(Berl),2011,56(3):159-165

    • [8] CHEN W,HAO Q Q,REN L L,et al.Cochlear morpholo⁃ gy in the developing inner ear of the porcine model of spontaneous deafness[J].BMC Neurosci,2018,19(1):28

    • [9] HEFFNER R S,HEFFNER H E.Hearing in domestic pigs(Sus scrofa)and goats(Capra hircus)[J].Hear Res,1990,48(3):231-240

    • [10] YI H J,GUO W,WU N,et al.The temporal bone micro⁃ dissection of miniature pigs as a useful large animal mod⁃ el for otologic research[J].Acta Otolaryngol,2014,134:26-33

    • [11] SHAKWEER W M E,HAFEZ Y M,El⁃Sayed A A,et al.Construction of ovine GH ⁃ pmKate2N expression vector and its uptake by ovine spermatozoa using different meth⁃ ods[J].J Genet Eng Biotechnol,2017,15(1):13-21

    • [12] 曾华沙,姚俊,王红顺,等.人/猪OSBPL2同源性比较及猪PFFs靶基因敲除细胞系的建立[J].南京医科大学学报(自然科学版),2018,38(2):149-154

    • [13] YAO J,ZENG H,ZHANG M,et al.OSBPL2 ⁃ disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia[J].J Genet Genom,2019,46(8):379-387

    • [14] BORNERT O,MØLLER T C,BOEUF J,et al.Identifica⁃ tion of a novel protein⁃protein interaction motif mediating interaction of GPCR ⁃ associated sorting proteins with G protein ⁃ coupled receptors[J].PLoS One,2013,8(2):e56336

    • [15] ABDULLAH N,BEG M,SOARES D,et al.Downregula⁃ tion of a GPCR by β⁃ arrestin2⁃mediated switch from an endosomal to a TGN recycling pathway[J].Cell Rep,2016,17(11):2966-2978

    • [16] 季丙元,陈京,白波.G蛋白偶联受体相关分选蛋白研究进展[J].济宁医学院学报,2013,36(4):287-291

    • [17] TUSZYNSKI G P,ROTHMAN V L,ZHENG X,et al.G ⁃ protein coupled receptor ⁃ associated sorting protein 1(GASP ⁃ 1),a potential biomarker in breast cancer[J].Exp Mol Pathol,2011,91(2):608-613

    • [18] RICKMAN D S,MILLON R,DE REYNIES A,et al.Pre⁃ diction of future metastasis and molecular characteriza⁃ tion of head and neck squamous⁃cell carcinoma based on transcriptome and genome analysis by microarrays[J].Oncogene,2008,27(51):6607-6622

    • [19] HORN S C,LALOWSKI M,GOEHLER H,et al.Hunting⁃ tin interacts with the receptor sorting family protein GASP2[J].J Neural Transm(Vienna),2006,113(8):1081-1090

    • [20] EDFAWY M,GUEDES J R,PEREIRA M I,et al.Abnor⁃ mal mGluR ⁃mediated synaptic plasticity and autism ⁃like behaviours in Gprasp2 mutant mice[J].Nat Commun,2019,10(1):1431

    • [21] HAI T,GUO W,YAO J,et al.Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis[J].Hum Genet,2017,136(11⁃12):1463-1475

  • 参考文献

    • [1] MYKYTYN K,ASKWITH C.G⁃protein⁃coupled receptor signaling in cilia[J].Cold Spring Harb Perspect Biol,2017,9(9):a028183

    • [2] ABU⁃HELO A,SIMONIN F.Identification and biological significance of G protein⁃coupled receptor associated sort⁃ ing proteins(GASPs)[J].Pharmacol Ther,2010,126(3):244-250

    • [3] XING G,YAO J,LIU C,et al.GPRASP2,a novel caus⁃ ative gene mutated in an X ⁃ linked recessive syndromic hearing loss[J].J Med Genet,2017,54:426-430

    • [4] STEPHANIE E,DIETMAR H,KATHARINA S,et al.Co⁃ chlea ⁃ specific deletion of Cav1.3 calcium channels ar⁃ rests inner hair cell differentiation and unravels pitfalls of conditional mouse models[J].Front Cell Neurosci,2019,13:225

    • [5] GUO W,YI H,REN L,et al.The morphology and electro⁃ physiology of the cochlea of the miniature pig[J].Anat Rec(Hoboken),2015,298:494-500

    • [6] 陈伟,陈磊,杨仕明,等.荣昌猪遗传性听力缺陷家系的发掘与应用[J].中华耳科学杂志,2016,14(1):10-14

    • [7] HOFFSTETTER M,LUGAUER F,KUNDU S,et al.Mid⁃ dle ear of human and pig:a comparison of structures and mechanics[J].Biomed Tech(Berl),2011,56(3):159-165

    • [8] CHEN W,HAO Q Q,REN L L,et al.Cochlear morpholo⁃ gy in the developing inner ear of the porcine model of spontaneous deafness[J].BMC Neurosci,2018,19(1):28

    • [9] HEFFNER R S,HEFFNER H E.Hearing in domestic pigs(Sus scrofa)and goats(Capra hircus)[J].Hear Res,1990,48(3):231-240

    • [10] YI H J,GUO W,WU N,et al.The temporal bone micro⁃ dissection of miniature pigs as a useful large animal mod⁃ el for otologic research[J].Acta Otolaryngol,2014,134:26-33

    • [11] SHAKWEER W M E,HAFEZ Y M,El⁃Sayed A A,et al.Construction of ovine GH ⁃ pmKate2N expression vector and its uptake by ovine spermatozoa using different meth⁃ ods[J].J Genet Eng Biotechnol,2017,15(1):13-21

    • [12] 曾华沙,姚俊,王红顺,等.人/猪OSBPL2同源性比较及猪PFFs靶基因敲除细胞系的建立[J].南京医科大学学报(自然科学版),2018,38(2):149-154

    • [13] YAO J,ZENG H,ZHANG M,et al.OSBPL2 ⁃ disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia[J].J Genet Genom,2019,46(8):379-387

    • [14] BORNERT O,MØLLER T C,BOEUF J,et al.Identifica⁃ tion of a novel protein⁃protein interaction motif mediating interaction of GPCR ⁃ associated sorting proteins with G protein ⁃ coupled receptors[J].PLoS One,2013,8(2):e56336

    • [15] ABDULLAH N,BEG M,SOARES D,et al.Downregula⁃ tion of a GPCR by β⁃ arrestin2⁃mediated switch from an endosomal to a TGN recycling pathway[J].Cell Rep,2016,17(11):2966-2978

    • [16] 季丙元,陈京,白波.G蛋白偶联受体相关分选蛋白研究进展[J].济宁医学院学报,2013,36(4):287-291

    • [17] TUSZYNSKI G P,ROTHMAN V L,ZHENG X,et al.G ⁃ protein coupled receptor ⁃ associated sorting protein 1(GASP ⁃ 1),a potential biomarker in breast cancer[J].Exp Mol Pathol,2011,91(2):608-613

    • [18] RICKMAN D S,MILLON R,DE REYNIES A,et al.Pre⁃ diction of future metastasis and molecular characteriza⁃ tion of head and neck squamous⁃cell carcinoma based on transcriptome and genome analysis by microarrays[J].Oncogene,2008,27(51):6607-6622

    • [19] HORN S C,LALOWSKI M,GOEHLER H,et al.Hunting⁃ tin interacts with the receptor sorting family protein GASP2[J].J Neural Transm(Vienna),2006,113(8):1081-1090

    • [20] EDFAWY M,GUEDES J R,PEREIRA M I,et al.Abnor⁃ mal mGluR ⁃mediated synaptic plasticity and autism ⁃like behaviours in Gprasp2 mutant mice[J].Nat Commun,2019,10(1):1431

    • [21] HAI T,GUO W,YAO J,et al.Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis[J].Hum Genet,2017,136(11⁃12):1463-1475