CN109247302A - 人雄激素受体复合物相关蛋白转基因小鼠 - Google Patents
人雄激素受体复合物相关蛋白转基因小鼠 Download PDFInfo
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Abstract
本发明涉及一种非人转基因动物,其在其基因组中含有可操作地连接至肝特异性启动子的编码人ARCAP的核酸。该转基因动物表达人ARCAP蛋白并且发生肝、脾、腹部或淋巴的肿瘤。也提供一种来自表达人ARCAP基因的非人转基因动物的细胞系。进一步提供一种产生转基因小鼠的方法,其通过以下方式进行:向受精小鼠卵母细胞中显微注射含有可操作地连接至肝特异性启动子的人ARCAP cDNA的载体,以及将经显微注射的该小鼠卵母细胞转移至培养小鼠中。
Description
技术领域
本发明涉及人雄激素受体复合物相关蛋白(ARCAP)转基因小鼠。
背景技术
发明背景
人雄激素受体复合物相关蛋白(Androgen Receptor Complex AssociatedProtein,ARCAP)基因最初是使用雄激素受体配体结合结构域作为诱饵,经由酵母双杂交分析分离的。人ARCAP mRNA具有2583个核苷酸的开放阅读框架(open reading frame),该开放阅读框架编码经计算的分子量为95Kd的860个氨基酸。参见美国专利6974683及7083935以及NCBI登录编号DQ768089。人ARCAP基因的分析揭露了其在染色体1q23.2-q24.3区处在135kb基因组DNA内包括19个外显子。
研究表明,人ARCAP基因在肝细胞瘤细胞系中表达,但在正常人肝细胞中不表达。参见美国专利6974683及7083935。
目前尚不了解ARCAP在肝细胞瘤发生中的作用。
肝细胞瘤的形成是一个复杂的过程。需要开发用于研究导致正常肝组织转变为肝肿瘤的形态学及分子病变的动物模型。
发明内容
发明概要
为了满足上述需要,本文提供一种非人转基因动物,其在其基因组中含有可操作地连接至肝特异性启动子的编码人ARCAP的核酸。该转基因动物表达人ARCAP蛋白并且发生肝、脾、腹部或淋巴的肿瘤。
也提供一种来自表达人ARCAP基因的非人转基因动物的细胞系。
另外,公开一种产生转基因小鼠的方法。该方法包括(i)向受精小鼠卵母细胞中显微注射含有可操作地连接至肝特异性启动子的人ARCAPcDNA的载体,以及(ii)将经显微注射的小鼠卵母细胞转移至培养小鼠(foster mouse)中以产生表达人ARCAP的转基因小鼠。该转基因小鼠发生肝、脾、腹部或淋巴肿瘤。
一个或多个实施方案的细节阐述于下文描述及实例中。其他特征、目的及优点将自数个实施方案的详细描述以及自申请专利范围显而易见。本文中所引用的所有公开案及专利文献均以全文引用的方式并入。
附图说明
以下描述参考附图,其中:
图1A展示人组织中的人ARCAP mRNA的Northern杂交分析的放射自显影图;
图1B展示所指示细胞系中的人ARCAP mRNA的Northern杂交分析的放射自显影图。A2、G2、22T、Hep3B、Sk-Hep-1为人肝细胞瘤细胞系,PC-3为人前列腺癌细胞系,且293T为永生化人胚肾细胞系。磷酸甘油醛脱氢酶(GAPDH)的表达被测量以作为内部对照;
图1C展示来自人肝肿瘤组织(T)及邻近正常组织(A)的成对组织中的人ARCAPmRNA及转铁蛋白对照的Northern杂交分析。
图2A为用于产生人ARCAP转基因动物的质体构建体的图。该构建体包括鼠白蛋白启动子及人ARCAP cDNA;
图2B为用于产生人ARCAP转基因动物的替代质体构建体的图。该构建体包括鼠磷酸烯醇丙酮酸羧激酶(PEPCK)启动子及人ARCAP cDNA;以及
图3展示人ARCAP蛋白(H;SEQ ID NO:2)及鼠ARCAP蛋白(M;SEQ ID NO:4)的氨基酸序列之间的比对。
具体实施方式
详细描述
如上所述,本文公开了表达人ARCAP的非人转基因动物。非人转基因动物可以为哺乳动物。举例而言,非人转基因动物可以为灵长类动物、有蹄动物、犬、鼠或猫。在一个具体实施方案中,非人转基因动物为小鼠。
转基因包括可操作地连接至肝特异性启动子的编码人ARCAP的核酸。肝特异性启动子可以为但不限于白蛋白启动子或磷酸烯醇丙酮酸羧激酶(phosphoenolpyruvatecarboxykinase,PEPCK)启动子。
编码人ARCAP蛋白的核酸可编码SEQ ID NO:2的氨基酸序列或与SEQ ID NO 2呈75%至95%(例如75%、80%、85%、90%、95%)一致的氨基酸序列。
鼠ARCAP蛋白(SEQ ID NO:4)与人ARCAP蛋白具有83%氨基酸一致性。参见图5。编码鼠ARCAP蛋白的核酸(参见例如SEQ ID NO:3)不属于本申请案的范畴。
编码人ARCAP蛋白的核酸可以包括SEQ ID NO:1的序列或与SEQ ID NO:1呈75%至99%一致的序列。在一个具体实施方案中,编码人ARCAP的核酸包括SEQ ID NO:1的序列。
上文所述的转基因动物表达人ARCAP蛋白并且发生肝、脾、腹部或淋巴的肿瘤。可在这些动物大约四个月大时在病理学上鉴别出肿瘤。
与表达人ARCAP蛋白的转基因小鼠不同,表达鼠ARCAP蛋白的转基因小鼠不会发生肿瘤。
可以自任何组织分离来自上文描述的非人转基因动物的细胞系。在某些实施方案中,细胞系来自肝、脾、腹部或淋巴肿瘤。
此外,上述用于产生转基因小鼠的方法包括向受精小鼠卵母细胞中显微注射含有可操作地连接至肝特异性启动子的人ARCAP cDNA的载体的步骤。
人ARCAP cDNA可以编码SEQ ID NO:2的氨基酸序列或与SEQ ID NO 2呈75%至95%(例如,75%、80%、85%、90%、95%)一致的氨基酸序列。同样,编码鼠ARCAP蛋白(SEQID NO:4)的核酸不属于本申请案的范畴。
人ARCAP cDNA可以包括SEQ ID NO:1的序列或与SEQ ID NO:1呈75%至99%一致的序列。在一个具体实施方案中,人ARCAP cDNA包括SEQ ID NO:1的序列。
人ARCAP cDNA可操作地连接至肝特异性启动子,例如鼠白蛋白启动子或鼠PEPCK启动子。
将经显微注射的小鼠卵母细胞转移至培养小鼠中,从而产生表达人ARCAP的转基因小鼠。该转基因小鼠发生肝、脾、腹部或淋巴肿瘤。
同样,表达鼠ARCAP蛋白的转基因小鼠并不发生肿瘤。
无需进一步详细描述,本领域技术人员可基于本文中的公开内容最大程度地利用本公开内容。因此,以下具体实例应理解为仅为说明性且无论如何不以任何方式限制本公开内容的其余部分。
实施例
实施例1:人ARCAP的表达
进行Northern杂交分析以评估人ARCAP mRNA在组织中的表达。结果展示在图1A中。人ARCAP mRNA在心及骨胳肌中弱表达。其不在大多个正常人组织中表达,包括正常肝组织。
在培养的肝细胞瘤细胞系中检查人ARCAP mRNA表达。结果展示在图1B中,表明ARCAP在人肝细胞瘤细胞系中高度表达,但在前列腺癌细胞或肾细胞中不表达。
也在来自罹患肝细胞癌的患者的成对组织样品中检查人ARCAPmRNA的表达。结果展示在图1C中。与邻近正常肝组织相比,在测试的15个肝细胞癌组织样品中的13个中,人ARCAP的表达量显著较高。此数据表明人ARCAP在肿瘤发生中起作用,特别在肝中。
实施例2:人ARCAP的过度表达在正常肝细胞中诱导癌症表型
正常肝细胞的永久性转染
将2.8kb全长人ARCAP cDNA克隆至载体pLXSN中并用于转染正常鼠肝BNL细胞。简而言之,BNL细胞在37℃下在10%CO2中生长。将与Lipofectamine 2000及培养基混合的质体DNA构建体在达至60-70%汇合(confluence)时加入细胞中。24小时后,用新鲜培养基更换该培养基。将遗传霉素(G418)添加至培养基中以选择稳定的转染体。用pLXSN-ARCAP转染BNL细胞,并用G418选择以建立过度表达人ARCAP的永久性转染细胞(BNL-ARCAP)。用空质体pLXSN转染对照细胞,并进行选择以产生BNL-pLXSN细胞。评估人ARCAP在BNL细胞中的表达对细胞迁移、细胞侵袭及固着非依赖性克隆生长(anchorage-independent clonalgrowth)的影响。
活体外创伤愈合分析
活体外创伤愈合分析用于测量组织培养物表面上的细胞迁移速率。此分析法模拟活体内创伤愈合期间的细胞迁移。其尤其适用于研究细胞-基质及细胞-细胞相互作用对细胞迁移的影响。
BNL细胞、BNL-pLXSN细胞及BNL-ARCAP细胞各自在组织培养瓶中以单层生长至汇合。用200μl移液管尖端轻轻地且缓慢地刮擦单层的表面以形成无细胞区域。在显微镜下观察细胞生长及向无细胞区域的迁移,并定期拍摄相片。使用影像分析软件(Image J)测量无细胞区域的大小。结果展示在下表1中。
表1.创伤愈合分析
细胞 | 6h<sup>a</sup> | 24h | 48h |
BNL | 0.6<sup>b</sup> | 0.4 | 0.2 |
BNL-pLXSN | 0.6 | 0.4 | 0.2 |
BNL-ARCAP | 0.4 | 0.2 | 0.1 |
a刮擦细胞单层后经过之时间
b无细胞区域的大小的倍数变化。BNL-ARCAP细胞的所有值与BNL及BNL-pLXSN两种细胞的相应值显著不同,p<0.05。
与未转染的BNL细胞或BNL-pLXSN细胞相比,过度表达人ARCAP的BNL细胞更快速且完全填充在无细胞区域中。
细胞迁移分析
通过用聚-L-赖氨酸涂布上部透孔(trans-well)来制备透孔(trans-well)组织培养室。将BNL、BNL-pLXSN及BNL-ARCAP细胞以每孔1×105个细胞的细胞密度接种至分别的上部透孔(trans-well)中。将透孔(trans-well)室在37℃下在10%CO2中培育。
细胞生长24小时后,自上部透孔(trans-well)中移除培养基。将保留在上部孔中的细胞及迁移至底部孔的细胞在室温下用4%多聚甲醛固定10分钟。所固定细胞用PBS冲洗3次,且接着与100%甲醇一起培育10分钟。用PBS洗涤后,加入0.05%结晶紫(CrystalViolet)持续10分钟以使细胞染色。经由显微镜观察上部及下部透孔(trans-well)中的细胞数并计数。结果展示在下表2中。
表2.透孔(Trans-well)细胞迁移分析
a值为在下部透孔(trans-well)中计数的细胞数
b与平均值的标准偏差
cBNL相对于BNL-pLXSN无显著不同,p=0.128
dBNL相对于BNL-ARCAP显著不同,p=0.0066
与BNL及BNL-pLXSN细胞相比,BNL-ARCAP细胞展示出显著更多的细胞跨越透孔(trans-well)迁移。
软琼脂集落形成分析
软琼脂集落形成分析用于定量活体外固着非依赖性细胞生长。细胞的固着非依赖性生长与其致瘤潜力相关。
按照标准程序用基础琼脂及顶层琼脂制备组织培养盘(plate)的个别孔(well)。在培养基中用1×104个细胞(HepG2、BNL、BNL-pLXSN及BNL-ARCAP)接种各孔,并置于在37℃下在10%CO2中的培养箱中。培养基每三天更换,持续21-28天。各孔中的细胞集落用0.005%结晶紫染色并用PBS洗涤。利用显微镜观察细胞集落并计数。结果展示在下表3中。
表3.软琼脂集落形成分析
a值为所形成的集落数
b与平均值的标准偏差
cBNL相对于BNL-pLXSN显著不同,p=0.025
dBNL相对于BNL-ARCAP显著不同,p=6.57×10-5
BNL-ARCAP细胞在所测试的细胞当中显示出最高程度的固着非依赖性生长。在分析中测试的HepG2人肝细胞瘤细胞形成如BNL-ARCAP细胞所形成的大约三分的二的集落数。在此分析中,对照BNL及BNL-pLXSN细胞不形成大量集落。
实施例3:转基因构建体
使用标准技术将2.8Kb全长人ARCAP cDNA次克隆至表达载体中的小鼠白蛋白启动子下游。参见图2A。在显微注射之前,用Not I及Sap I消化所得的5.8Kb白蛋白启动子/ARCAP构建体。
也将ARCAP cDNA克隆至表达载体中的小鼠PEPCK启动子下游。参见图2B。在显微注射之前,用Asc I消化所得的5.7Kb PEPCK启动子/ARCAP构建体。
实施例4:原核显微注射
在台湾动物中心(Taiwan Animal Center,台湾台北(Taipei,Taiwan))将含有ARCAP的DNA显微注射至受精的C57/BL6J雌性胚胎(0.5dpc胚胎)中。根据标准方案将注射的受精卵转移回培养小鼠母亲中。产生转基因小鼠并在标准的无特定病原体条件下饲养。所有动物研究均按照台北荣民总医院(Taipei-Veterans General Hospital)动物设施研究所的实验动物照护及使用委员会(Institutional Animal Care and Use Committee)制定的规则进行。
实施例5:表达hARCAP的转基因小鼠发生肿瘤
对20只自白蛋白启动子表达人ARCAP的转基因小鼠及20只自白蛋白启动子表达人ARCAP的小鼠进行分析。所有40只小鼠在出生后3个月内均发生肿瘤。
重要的是,各自在肝特异性启动子控制下过度表达鼠ARCAP基因的10只转基因小鼠已经无肿瘤长达两年。
对自正常肝、异常肝、肝肿瘤、腹部肿瘤、正常血液、异常血液、正常脾、增大的脾及脾肿瘤中提取的人ARCAP转基因小鼠RNA样品进行RT-PCR分析。正常组织呈现出在组织学上正常。结果展示在下表4中。异常组织展示出病理改变,但没有明显的肿瘤。举例而言,正常肝展示出预期的肝组织学。异常肝显示出颜色异常,轻微增大以及出现结节。利用组织学证实肿瘤样品为恶性的。
表4.人ARCAP转基因小鼠组织中的相对mRNA表达
a脾组织RNA表达量是相对于正常脾
*与正常肝或正常脾显著不同,p<0.05
数据展现出,ARCAP在白蛋白启动子/人ARCAP转基因小鼠的肝、脾及腹部肿瘤中高度表达。也观察到细胞角蛋白19(CK19;细胞增殖标志物)、酪氨酸氨基转移酶(TAT;与肝炎相关)及甲胎蛋白(AFP;肝癌标志物)的表达升高。参见上文表4。
在PEPCK启动子/ARCAP转基因小鼠中,RT-PCR分析也显示脾肿瘤组织中高量的ARCAP表达。参见同上。也注意到高量的CDK19、TAT及AFP,尤其在腹部肿瘤中。
此外,所有人ARCAP转基因小鼠均没有B型肝炎病毒及C型肝炎病毒感染。
其他实施方案
本说明书中公开的所有特征可以任何组合形式组合。本说明书中公开的各特征可经用于相同、等效或类似目的的替代性特征置换。因此,除非另外明确说明,否则所公开的各特征仅为一系列通用等效或类似特征的一个实例。
根据以上描述,本领域技术人员可容易地确定本公开内容的基本特征,且在不脱离本公开内容的精神及范畴的情况下可对本公开内容作出各种改变及修改以使其适应各种用途及条件。因此,其他实施方案也在申请专利范围内。
Claims (17)
1.一种非人转基因动物,其在其基因组中包含可操作地连接至肝特异性启动子的编码人雄激素受体复合物相关蛋白(ARCAP)的核酸,其中该转基因动物表达人ARCAP蛋白并且发生肝、脾、腹部或淋巴肿瘤。
2.如权利要求1的非人转基因动物,其中该非人转基因动物为哺乳动物。
3.如权利要求2的非人转基因动物,其中该哺乳动物为灵长类动物、有蹄动物、犬、鼠或猫。
4.如权利要求3的非人转基因动物,其中该哺乳动物为小鼠。
5.如权利要求1的非人转基因动物,其中该肝特异性启动子为白蛋白启动子或磷酸烯醇丙酮酸羧激酶(PEPCK)启动子。
6.如权利要求4的非人转基因动物,其中该肝特异性启动子为鼠白蛋白启动子或鼠磷酸烯醇丙酮酸羧激酶(PEPCK)启动子。
7.如权利要求4的非人转基因动物,其中该核酸包括SEQ ID NO:1的序列。
8.如权利要求6的非人转基因动物,其中该核酸包括SEQ ID NO:1的序列。
9.一种细胞系,其来自如权利要求1的非人转基因动物。
10.如权利要求9的细胞系,其中该细胞系来自该肝、脾、腹部或淋巴肿瘤。
11.一种细胞系,其来自如权利要求4的非人转基因动物。
12.如权利要求11的细胞系,其中该细胞系来自该肝、脾、腹部或淋巴肿瘤。
13.一种细胞系,其来自如权利要求6的非人转基因动物。
14.如权利要求13的细胞系,其中该细胞系来自该肝、脾、腹部或淋巴肿瘤。
15.一种用于产生转基因小鼠的方法,该方法包含:
向受精小鼠卵母细胞中显微注射含有可操作地连接至肝特异性启动子的人雄激素受体复合物相关蛋白(ARCAP)cDNA的载体,以及
将经显微注射的该小鼠卵母细胞转移至培养小鼠中,从而产生表达人ARCAP的转基因小鼠,
其中该转基因小鼠发生肝、脾、腹部或淋巴肿瘤。
16.如权利要求15的方法,其中该肝特异性启动子为鼠白蛋白启动子或鼠磷酸烯醇丙酮酸羧激酶(PEPCK)启动子。
17.如权利要求16的方法,其中该cDNA包括SEQ ID NO:1的序列。
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US5907078A (en) * | 1994-12-09 | 1999-05-25 | Greenberg; Norman M. | Transgenic mouse model for prostate cancer |
CN1375503A (zh) * | 2001-01-17 | 2002-10-23 | 台北荣民总医院 | 雄激素受体复合物相关蛋白 |
WO2004007753A2 (en) * | 2002-07-17 | 2004-01-22 | Bristol-Myers Squibb Company | Transgenic non-human mammals expressing a reporter nucleic acid under the regulation of androgen response elements |
CN1495195A (zh) * | 2002-07-25 | 2004-05-12 | 台北荣民总医院 | 表达雄激素受体复合物相关蛋白的转基因动物 |
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WO2007025269A2 (en) * | 2005-08-25 | 2007-03-01 | Michigan State University | Transgenic mouse model and methods for treatment of neuro muscular disease by interfering with androgen-androgen receptor interaction in skeletal muscles |
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