CN114586735B - Pparg基因定点突变小鼠模型的构建与应用 - Google Patents
Pparg基因定点突变小鼠模型的构建与应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,涉及两种Pparg基因定点突变小鼠模型的构建和应用。本发明运用CRISPR/Cas9基因编辑技术,对Pparg基因的编码第166位苏氨酸的位置进行人工突变,并以此成功制作了两种定点突变小鼠品系。本发明亦基于获得的Pparg基因定点突变小鼠品系,通过多种细胞、生化、分子生物学的研究手段,分析了这两种品系的多种生理与病理状态、确定了其用于相关研究的应用价值。同时,Pparg基因定点突变小鼠可用于相关药物的筛选、药物设计、药理/毒理学、药效学、药物代谢动力学以及治疗靶标发掘的应用。
Description
技术领域
本发明属于基因工程技术领域,涉及两种Pparg基因定点突变小鼠模型的构建和应用。
背景技术
PPARγ是一种在细胞增殖、分化、代谢及功能表型维持等诸多生物学过程中具有重要地位的核受体转录因子。PPARγ存在于多种细胞类型中,如脂肪细胞、肝脏细胞、肌肉细胞等代谢器官来源细胞;巨噬细胞、T/B细胞、树突状细胞等免疫细胞;神经元等神经系统来源的细胞中。作为经典的临床药物靶点,PPARγ在受到完全激动剂噻唑烷二酮类(TZDs)药物激动的情况下,可非常显著的增强II型糖尿病患者的胰岛素敏感性,降低血糖。同时,这种激动作用可以降低代谢紊乱的器官及组织中的炎性细胞的浸润,增强免疫调控性细胞的活力,以此维持机体的代谢和免疫稳态。但PPARγ活力的完全激活也会带来诸多不利的副作用,这背后的本质因素是由于我们对PPARγ广泛的生物学行为以及其作用机制的研究存在大量的空白。同时,围绕该靶点的药物开发亦陷入停滞阶段。
转基因小鼠模型对对生命科学、基础医学的发展具有里程碑式的意义,对临床疾病的认识和治疗也起到了极大的推动作用。相比于基因敲除小鼠,点突变的小鼠在研究生命过程及药物开发中具有更强的优势。首先,点突变小鼠在不丧失蛋白质组分的情况下对靶蛋白的功能进行了直接调控;其次,点突变小鼠可展示蛋白质结构位点的精细变化所带来的蛋白功能,乃至细胞生理、病理结构变化的趋势;最后,点突变小鼠可以明确药物对靶蛋白调控的作用机理,提供药物分子与蛋白质结合位点之间的关联信息。综上述,点突变小鼠可为研究蛋白新功能,理解生命过程,并以此开发新型的药物提供思路。
到目前为止,Pparg基因定点突变小鼠的开发存在大量空白。基于前期的研究工作基础,本专利报道了两种Pparg基因定点突变小鼠的构建方法与应用。这两种转基因小鼠品系的建立为研究PPARγ生物学及其相关的代谢、免疫与神经系统的生理病理过程提供了新的模式动物。同时,这两种动物模型在围绕PPARγ本体,或其翻译后修饰,或其基因多态性的药物研发中亦有重要的作用。
发明内容
本发明第一目的在于提供一种Pparg基因定点突变小鼠模型的构建方法,用来对Pparg基因编码第166位苏氨酸的位置进行人工突变,并以此成功制作了两种品系全身性的定点突变小鼠。这两个品系分别为PPARγ第166位苏氨酸突变为丙氨酸(T166A突变,简称TA突变)小鼠,及PPARγ第166位苏氨酸突变为天冬氨酸(T166D突变,简称TD突变)小鼠(166位苏氨酸为Pparg基因编码的最长转录本翻译后产生的PPARγ2蛋白对应位置的氨基酸,在PPARγ1中为136位苏氨酸。无论PPARγ1或PPARγ2,皆由同一Pparg基因编码)。
本发明的第二目的在于提供Pparg基因定点突变小鼠作为模型在研究生理与病理过程中的应用。
本发明的第三目的在于提供Pparg基因定点突变小鼠模型在药物筛选、药物设计及开发中的应用。
本发明的第四目的在于提供Pparg基因定点突变小鼠模型在药理学、药效学、药物代谢动力学以及诊断与治疗靶标发掘研究中的应用。
本发明的第五目的在于提供Pparg基因定点突变小鼠模型用于开发制备检测试剂盒、试纸或芯片中的应用。
所述的转基因小鼠构建方法如下:
(1)设计高效的识别特定基因组PAM区域的gRNA序列;
(2)构建Cas9打靶载体和donor载体;
(3)将Cas9打靶载体、gRNA及donor载体显微注射到C57BL/6J小鼠的受精卵进行同源重组,并移植给代孕母鼠,获得F0代小鼠;
(4)经PCR和测序验证阳性的F0代小鼠与C57BL/6J小鼠交配获得可稳定遗传的F1代小鼠模型。
(5)经PCR和测序验证阳性的F1代小鼠进行杂交,利用PCR测序筛选纯合子突变小鼠。
以上构建方法为一个具体的实施实例,所述的定点突变引入是采用本领域技术人员所掌握的方法,但不限于同源重组、锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALENs)以及CRISPR/Cas9系统。
在具体实施实例中,所述的CRISPR/Cas9系统中的单链引导gRNA序列如序列表SEQID NO.1与NO.2所示;引入TA突变或TD突变的Donor DNA序列如序列表SEQ ID NO.3(TA突变)与NO.4(TD突变)所示。
所述的Pparg基因定点突变小鼠作为模型在研究生理与病理过程中的应用。根据PPARγ对内分泌系统、免疫系统及神经系统广泛的调控功能,因此应用于研究的生理过程包括:物质与能量代谢、代谢器官或组织的发育及运作、内分泌功能、细胞代谢功能与机理等代谢内分泌相关的生理功能;免疫细胞发育与活化、免疫器官的发育与运作、血细胞发育与功能、免疫应答及调控、及肿瘤免疫等免疫系统相关的生理功能;神经细胞发育及功能、神经系统调节机能、及神经信号传导等神经系统相关生理功能。
在一个具体的实施实例中,TA与TD小鼠展现出独特的生理病理表型,包括脂肪组织、肝脏、肌肉等代谢器官的组织结构变化;从TA与TD小鼠体内分选的细胞具特征性的代谢表型;TA与TD小鼠在物质与能量代谢、胰岛素敏感性等代谢内分泌相关的生理功能方面各自具有独特性。该实例体现了TA与TD小鼠在代谢及内分泌系统生理病理研究中的应用。
在一个具体的实施实例中,TA与TD小鼠来源的免疫细胞,特别是巨噬细胞的极化方式、吞噬功能等皆有各自特征的表型。该实例体现TA与TD小鼠在免疫系统生理功功能研究中的应用。
在一个具体的实施实例中,TA与TD小鼠来源的神经元细胞,神经信号传导及其对外部组织控制的能力发生了变化。该实例体现TA与TD小鼠在神经系统生理功利研究中的应用。
所述的病理过程皆与PPARγ功能紧密相连,具体疾病类型包括代谢性疾病:肥胖、糖尿病、脂肪组织代谢紊乱、脂肪细胞分化障碍、动脉粥样硬化、肿瘤、代谢性肾脏疾病、代谢性肝病、代谢性肌肉疾病等。亦包含PPARγ蛋白功能异常所导致的免疫系统疾病:代谢性炎症、急性与慢性炎症、免疫细胞或系统发育不良、自体免疫疾病、过敏及肿瘤免疫异常等。
所述的Pparg基因定点突变小鼠模型在药物筛选、药物设计及开发中的应用。具体包括:运用TA与TD小鼠活体,或利用其分离的细胞组织样本,用于[0017]中所述的任意一种疾病的药物筛选、设计、优化及相应制剂制备。药物的类型包括:调控PPARγ功能或活性的小分子化合物、短肽、抗体或酶;与PPARγ功能相关的蛋白、质粒载体;调控PPARγ功能或活性基因编辑工具及病毒;免疫细胞制剂、干细胞制剂及其它细胞疗法制剂。
在一个具体的实施实例中,运用Pparg基因定点突变小鼠模型筛选获得了一系列可干预PPARγ活性与功能的配体小分子化合物;进一步运用Pparg基因定点突变小鼠模型评估了化合物对代谢系统,免疫系统等系统的作用;通过提取Pparg基因定点突变小鼠的细胞,建立体外培养模型,可用于研究并评估药物对细胞生命的调控作用。该实例体现TA与TD小鼠在药物筛选、药物设计及开发中的应用。
所述的Pparg基因定点突变小鼠模型在药理/毒理学、药效学、药物代谢动力学以及诊断与治疗靶标发掘研究中的应用。具体包括:运用TA与TD小鼠活体,或利用其分离的细胞组织样本,用于[0013]与[0017]中所述的任意一种生理或病理过程相关的药理学、药效学以及诊断治疗靶标的发掘研究。亦包括运用运用TA与TD小鼠活体,进行相关药物的药物代谢动力学的研究。
在一个具体的实施实例中,运用Pparg基因定点突变小鼠模型评估了胰岛素增敏剂对代谢系统,免疫系统等系统的作用;通过提取Pparg基因定点突变小鼠的细胞,建立体外培养模型,研究并评估了相关药物分子的作用机理;亦通过药物代谢的研究方法,评估了药物在Pparg基因定点突变小鼠模型中的药物分布特定以及药物毒性作用。该实例体现TA与TD小鼠在药理学、药效学、药物代谢动力学以及诊断与治疗靶标发掘研究中的应用。
所述的Pparg基因定点突变小鼠模型用于开发制备检测试剂盒、试纸或芯片中的应用。具体包括:运用TA与TD小鼠分离的细胞组织样本、细胞组织提取物或体液等制备用于检测ELISA、Western blotting、免疫组织化学(IHC)及免疫荧光(IF)等免疫检测试剂;组织芯片或基因芯片;识别PPARγ及其相关突变体蛋白、翻译后修饰蛋白等的抗体试剂生产。
在一个具体的实施实例中,提取Pparg基因定点突变小鼠的细胞并制备为细胞裂解物,通过Western blotting检测了PPARγ的突变体蛋白;同时,将Pparg基因定点突变小鼠的组织制备为石蜡切片,可用于免疫组化分析。该实例体现TA与TD小鼠在开发制备检测试剂盒、试纸或芯片中的应用。
附图说明
图1为Pparg基因定点突变小鼠构建策略示意图。
图2为使用PCR发对纯合子小鼠进行鉴定的电泳示意图。
图3为使用病理切片分析对Pparg基因定点突变小鼠脂肪组织的形态特征进行评估;使用荧光定量PCR技术对脂肪组织中脂质代谢相关基因的表达进行分析。
图4为在高脂饲喂的条件下,对Pparg基因定点突变小鼠的肝脏和肌肉组织进行病理切片分析示意图。
图5为使用病理切片分析对高脂饲喂状态下Pparg基因定点突变小鼠脂肪组织的形态特征与促炎性免疫细胞浸润情况;使用荧光定量PCR对Pparg基因定点突变小鼠巨噬细胞表型进行检测。
图6为高脂饲喂状态下测定Pparg基因定点突变小鼠胰岛素敏感性状态(图6.B-C),以及测定血清中代谢指标物变化(图6.D-E)。
图7为在冷刺激状态下,使用热成像仪对Pparg基因定点突变小鼠的体温进行监测(图7.A);同时在体外建立细胞培养模型,使用神经递质激动剂异丙肾上腺素刺激细胞,定量PCR测定相关基因表达(图7.B)。
图8为运用Pparg基因定点突变小鼠进行药物代谢脑分布的评估(图8.A),以及肠道和骨骼毒副作用的评估(图8.B-C)。
图9为使用病理切片技术、免疫组化技术分析Pparg基因定点突变小鼠的脂肪组织形态结构及代谢标志物表达与分布状态(图9.A-B);同时,Pparg基因定点突变小鼠细胞裂解液可应用于Western blotting技术的检测。
图10为使用PPARγ的配体激动剂罗格列酮(Rosiglitazone,RSG)对Pparg基因定点突变小鼠进行给药处理,评估药理及药效,分析该靶点的激动对代谢系统的影响。
图11为Pparg基因定点突变小鼠的细胞可用于免疫荧光相关的分析检测(图11.A),也可用于基因芯片及测序相关的产品的开发(图11.B)。
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并未用于限定本发明的范围。
以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例及附图来详细说明本发明。
实施实例一:Pparg基因定点突变小鼠模型的构建方法,原理如图1所示,具体地包括如下步骤:
1.Pparg基因CRISPR/Cas9 gRNA的设计:
根据Pparg基因序列和需要引入的突变T166A(ACC→GCT)、T166D(ACC→GAT),设计两条gRNA序列,具体序列见序列表SEQ ID NO.1(T166A突变)与NO.2(T166D突变)
2.Donor载体的构建:
Donor载体构建方法是先人工合成5′端带有不同酶切位点识别序列的靶序列寡核苷酸引物,通过PCR对两对引物直接退火,合成出携带不同的黏性末端的靶序列DNA短片段,将其插入到载体中,构建小鼠靶向Pparg基因第166位苏氨酸对应编码序列的Donor载体(CRISPR/Cas9打靶载体),产生两个载体:Donor-T166A和Donor-T166D。Donor序列见序列表SEQ ID NO.3(T166A)与NO.4(T166D)。
具体步骤为:
(1)引物退火。寡聚核苷酸Pparg-T166A-F、Pparg-T166A-R直接退火,两条引物形成带有不同黏性末端的短片段,退火反应程序为90℃10min,70℃10min,自然冷却至室温。
(2)载体酶切。用内切酶对CRISPR/Cas9骨架载体进行切割。酶切产物用1%的琼脂胶检测,按照琼脂糖凝胶回收试剂盒说明书回收线性化的载体。
(3)链接反应。使用T4连接酶对线性化的载体和退火合成的Pparg-T166A或Pparg-T166D短片段连接,16℃连接12~16h。
(4)连接产物转化,按照常规方法进行。
(5)测序鉴定。随机选取2-3个单克隆菌落进行扩大培养,提取质粒,用U6引物进行测序鉴定,确保插入表达载体的DNA序列与设计的一致,最终判定成功获得CRISPR/Cas9-Pparg-T166A载体和CRISPR/Cas9-Pparg-T166D载体。
(3)Donor质粒的构建
在不改变C57小鼠其他基因碱基序列的情况下,参考上述基因信息以及gRNA活性靶点,制备Donor质粒。
3.显微注射
6~8周龄C57BL/6J雄性小鼠和雌性小鼠交配后,获取受精卵,将gRNA、donor载体及CRISPR/Cas9载体一并显微注射到受精卵中,另取5只同期输卵管结扎假孕母鼠作为受体,将注射后的受精卵转移植入假孕的母鼠输卵管中。
4.F0代小鼠鉴定
经过显微注射和胚胎移植后,F0代小鼠出生。经过PCR电泳图(图2.A-B)、测序鉴定,确定了基因型正确。
5.将通过上述构建方法得到的基因鼠纯合子分别进行如下测试。
实施实例二:8周大TA与TD小鼠进行脂肪组织切片及H&E染色,通过分析脂肪组织形态特征可以发现,TA小鼠的脂肪组织中的脂肪细胞较小,数量较多,单个脂肪细胞囤积的脂质较少;相反地,TD小鼠的脂肪细胞较大,数量少,单个脂肪细胞囤积的脂质较多(图3.A);同步地,运用荧光定量PCR分析脂肪组织中代谢相关基因的变化,可以发现TA小鼠的脂肪组织具有高脂肪酸代谢特征,而TD小鼠的脂肪酸分解代谢受阻(图3.B)。随后,对两种点突变小鼠进行高脂饲喂3个月,并分离肝脏和肌肉组织分析其病理状态,结果显示,相比于野生型小鼠,TA的肝脏和肌肉中具有显著降低的脂变性(图4.A-B)。同步分析胰岛素敏感性、物质与能量代谢状态等,皆显示TA小鼠具有更加健康的内分泌系统与代谢表型(图6.A-E)。该实例体现了TA与TD小鼠在代谢及内分泌系统生理病理研究中的应用。
实施实例三:对点突变小鼠与野生型小鼠进行3个月的高脂饲喂(60%脂肪含量饲料)。随后,分离小鼠内脏脂肪并进行石蜡切片,运用H&E病理染色法对组织切片进行分析;结果显示,野生型小鼠的内脏脂肪组织中浸润了大量的炎性免疫细胞,脂肪组织呈现慢性炎症状态,而TA小鼠的内脏脂肪组织中却含有较少的免疫细胞浸润(图5.A)。与此同时,分选小鼠原代的巨噬细胞,并进行定量PCR分析,可以发现TA突变会降低巨噬细胞经典活化(M1极化),增强巨噬细胞替代活化表型(M2极化)。相反地,TD突变会降低巨噬细胞替代活化表型(M2极化),反而增强经典活化(M1极化)(图5.B)。该实例体现TA与TD小鼠在免疫系统生理功功能研究中的应用。
实施实例四:运用冷刺激模型对TA与TD小鼠进行处理,通过热成像监控神经系统对体温的调节功能,结果显示,TD小鼠的体温低于TA与野生型小鼠,这说明TD突变对神经系统的体温调节功能产生了影响(图7.A)。另一方面,分离原代的小鼠脂肪细胞,在体外使用神经递质激动剂异丙肾上腺素(Isoprenaline,ISO)处理脂肪细胞,并使用荧光定量PCR检测神经递质对脂肪的调控功能,结果显示,TA小鼠能够更好的响应神经调控,而TD则对神经调节钝化(图7.B)。该实例体现TA与TD小鼠在神经系统生理功利研究中的应用。
实施实例五:运用TA与TD小鼠,腹腔注射PPARγ的小分子配体化合物,通过组织学分析,Western blotting检测、以及荧光定量PCR检测,可以发现,TA小鼠可以更好的响应小分子配体,上调脂肪组织脂肪酸氧化代谢与褐色化生物标志物;与之相反地,TD小鼠不响应这些小分子配体,呈现脂肪合成增加,脂肪酸氧化代谢减弱的表型。同时,通过从TA与TD小鼠中分选原代的脂肪细胞,体外使用PPARγ配体小分子进行处理,结果亦证明在体结论,即这些小分子是通过调控PPARγ的构象及生物学行为实现对细胞的功能性调控(图9-10)。该实例体现TA与TD小鼠在药物筛选、药物设计及开发中的应用。
实施实例六:在[0042]的基础上,运用TA与TD小鼠,腹腔注射PPARγ的小分子配体化合物,经过14天的给药后,腹腔注射2%Evans blue染料,分析血脑屏障的完整性,以此评估两种化合物的脑部药物分布特点(图8.A);为评估对消化道的副作用,记录了小鼠的摄食情况(图8.B);为评估药物对骨密度的影响,使用X-RAY成像发分析小鼠BMD指数,发现TA小鼠在接受药物后,骨密度降低,说明该小鼠对促骨质疏松药物敏感,即该模型可用于评估药物的骨相关毒副作用(图8.C)。该实例体现TA与TD小鼠在药理学、药效学、药物代谢动力学以及诊断与治疗靶标发掘研究中的应用。
实施实例七:如图9所示,点突变小鼠的细胞提取物可用于Western blotting检测;石蜡切片可用于免疫组化分析。此外,如图11所述,点突变小鼠分选的细胞可用于免疫荧光分析及基因芯片、测序等高通量分析。该实例体现TA与TD小鼠在开发制备检测试剂盒、试纸或芯片中的应用。
以上所述实施例仅表达了本发明的几种实施方式,但并不能因此而理解为对发明专利范围的限制。以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (3)
1.一种Pparg基因定点突变小鼠模型的构建方法,其特征在于,包括以下步骤:
第1步:设计识别特定基因组PAM区域的gRNA序列,其序列如序列表SEQ ID No.1所示;
第2步:构建Cas9打靶载体和donor载体,Donor DNA序列如SEQ ID NO.3所示;
第3步:将Cas9打靶载体、gRNA及donor载体显微注射到C57BL/6J小鼠的受精卵进行同源重组,并移植给代孕母鼠,获得F0代小鼠;
第4步:经PCR和测序验证阳性的F0代小鼠与C57BL/6J小鼠交配获得可稳定遗传的F1代小鼠模型。
第5步:经PCR和测序验证阳性的F1代小鼠进行杂交,利用PCR测序筛选纯合子突变小鼠。
2.一种Pparg基因定点突变小鼠模型的构建方法,其特征在于,包括以下步骤:
第1步:设计识别特定基因组PAM区域的gRNA序列,其序列如序列表SEQ ID No.2所示;
第2步:构建Cas9打靶载体和donor载体,Donor DNA序列如SEQ ID NO.4所示;
第3步:将Cas9打靶载体、gRNA及donor载体显微注射到C57BL/6J小鼠的受精卵进行同源重组,并移植给代孕母鼠,获得F0代小鼠;
第4步:经PCR和测序验证阳性的F0代小鼠与C57BL/6J小鼠交配获得可稳定遗传的F1代小鼠模型。
第5步:经PCR和测序验证阳性的F1代小鼠进行杂交,利用PCR测序筛选纯合子突变小鼠。
3.权利要求1或2所述Pparg基因定点突变小鼠模型的构建方法得到的小鼠模型在研究脂肪酸代谢相关生理与病理过程中的应用。
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