CN102971420A - 无毛nod scid小鼠 - Google Patents
无毛nod scid小鼠 Download PDFInfo
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- CN102971420A CN102971420A CN2011800291571A CN201180029157A CN102971420A CN 102971420 A CN102971420 A CN 102971420A CN 2011800291571 A CN2011800291571 A CN 2011800291571A CN 201180029157 A CN201180029157 A CN 201180029157A CN 102971420 A CN102971420 A CN 102971420A
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Abstract
本文公开了非肥胖糖尿病(NOD)背景下的无毛、免疫缺陷小鼠及其生产方法。所述小鼠为无毛的，并且具有多重免疫缺陷，包括B细胞和T细胞缺陷，以及受损的巨噬细胞和补体功能。所述小鼠还在免疫系统的自然杀伤细胞和树突细胞中具有进一步的缺陷。所述小鼠可用于生物医学研究，例如涉及异种移植、自发性肿瘤、癌细胞肿瘤发生、肿瘤血管发生、肿瘤转移潜能、肿瘤抑制治疗、致癌调控和肿瘤成像的研究。
Description
相关申请的交叉引用
本申请要求35 U.S.C. § 119(e) 下于2010年4月14日提交的美国临时申请序列号61/324,030和于2010年10月8日提交的美国临时申请序列号61/391,444的优先权，其各自的全部公开内容通过引用结合到本文中。
技术领域
本公开内容概括而言涉及生物医学研究。更具体地，本公开内容涉及具有用于生物医学研究的遗传优势的小鼠。
背景和简述
在小鼠和大鼠中有数以百计的病理学突变体，其中的许多是为了生物医学研究的目的。突变小鼠通常用于专门的研究和开发项目。突变小鼠可用远交或近交遗传背景维持，其性质赋予遗传优势或劣势。例如，远交HsdCpb:NMRI背景下的无胸腺裸基因产生相对强壮的动物，而在具有BALB/cOlaHsd背景下的小鼠中相同突变产生不太强壮的动物。
Prkdc scid 为编码DNA活化蛋白激酶(Prkdc)催化亚基的基因的突变，导致重度联合免疫缺陷(scid)(综述见Bosma和Carroll，1991)。其为常染色体隐性突变，由于产生B和T细胞受体所必需的可变、多样和连接的(VDJ)重组酶系统缺陷使B细胞和T细胞祖细胞分化中断。Prkdc突变的小鼠缺乏Thy-1阳性树突表皮细胞。与裸鼠不同的是，Prkdc scid 突变小鼠具有胸腺和淋巴结。
非肥胖糖尿病(NOD)小鼠为1型糖尿病动物模型，其显示对自身免疫性T细胞介导的胰岛素依赖性糖尿病(IDDM)自发发生的易感性。NOD品系和相关品系由Makino及其同事在日本Aburahi的Shionogi研究实验室开发，并且在1980年首次报道(Makino等，1980)。此外，NOD品系通常特征在于自然杀伤(NK)细胞功能缺陷、骨髓发育和功能以及抗原呈递细胞(APC)分化和功能缺陷、抑制溶血补体的经典和替代途径的C5缺陷(Greiner等，1998)。
携带Prkdc scid 突变的小鼠品系虽然显示B和T细胞功能缺乏，但表现出正常水平的NK细胞、溶血补体和骨髓功能。此外，随小鼠变老各种背景品系的Prkdc scid 突变体产生低水平的免疫球蛋白和功能性T细胞，这一现象被称为“渗漏(leakiness)”。这些先天免疫性质对某些研究应用，特别是人肿瘤细胞系的移入是不利的。
Prkdc scid 突变已经由C.B-17背景转移至糖尿病易感的非肥胖性糖尿病(NOD)背景，被称为NOD scid。与通常的NOD小鼠不同的是，自身免疫性糖尿病并非由于T细胞功能缺乏而发生。NOD scid小鼠的特征进一步在于NK细胞功能缺陷、巨噬细胞缺陷和可检测的溶血补体缺乏(Greiner等，1998)。
此外，与多种遗传背景下的其它scid小鼠相比，由VDJ重组酶活性的不完全阻断(其导致残留B和T细胞活性)引起的纯合scid小鼠的“渗漏”表型在本模型中被抑制。这些特征对涉及移植细胞的长期生长的研究是有利的。例如，人细胞系例如PC-3、DU145以及CD-34阳性干细胞可在NOD scid小鼠中有效地长期生长，但在NK细胞功能、巨噬细胞和循环补体(其可影响肿瘤生长或人干细胞移入)正常的其它scid小鼠中不能。NOD scid小鼠的免疫概况使其可用于肿瘤学、免疫学、血液学、HIV病理学和其它领域的生物医学研究。
尽管适合多数应用，但NOD scid小鼠显示的一些特征可对某些研究目的不利。例如，NOD scid小鼠具有白化毛的完全皮毛，因此在接种细胞前须将毛刮去，随后的肿瘤显示和成像比无毛模型困难。此外，甚至在刮去有毛小鼠的毛后，也残留毛囊，这可产生自身荧光，干扰使用生物发光和荧光报告物的成像研究。与裸鼠不同，无毛纯合突变体缺乏毛囊(Lyon等，1996)，因此消除了其它异种移植模型中出现的毛囊自身荧光。
本文描述了NOD scid小鼠的改进，其如下进行：将NOD scid小鼠与携带无毛基因(Hr)突变的小鼠杂交并与NOD scid遗传背景回交得到无毛NOD scid小鼠品系。本文公开的无毛NOD scid小鼠对异种移植、自发性肿瘤、癌细胞肿瘤发生、肿瘤血管发生、肿瘤转移潜能、肿瘤抑制治疗、致癌调控、肿瘤成像和人干细胞移入的研究尤其有利。
考虑下述多个实施方案：
1) 非肥胖糖尿病(NOD)背景下具有B细胞、T细胞、NK细胞、巨噬细胞和补体缺陷的无毛、免疫缺陷小鼠。
2) 条款1的小鼠，其中所述小鼠为Prkdc等位基因纯合的。
3) 条款2的小鼠，其中所述Prkdc等位基因为scid (Prkdc scid )。
4) 条款1-3中任一项的小鼠，其中所述小鼠为Hr等位基因纯合的。
5) 条款4的小鼠，其中所述Hr等位基因为hr (Hr hr )。
6) 条款1-5中任一项的小鼠，其中所述非肥胖糖尿病背景为NOD.CB17-Prkdc scid 。
7) 条款1-5中任一项的小鼠，其中非肥胖糖尿病背景为NOD.CB17-Prkdc scid /NCrHsd。
8) 条款1-7中任一项的无毛小鼠，进一步具有树突细胞缺陷，其中所述树突细胞缺陷为与相同背景的非无毛免疫缺陷小鼠相比树突细胞的数目减少。
9) 生产非肥胖糖尿病(NOD)背景下无毛、免疫缺陷小鼠的方法，所述方法包括：
(a) 将Prkdc等位基因纯合的NOD scid小鼠品系与Hr等位基因纯合的第二小鼠品系杂交，以产生Hr等位基因和Prkdc等位基因均杂合的子代；
(b) 将步骤(a)产生的杂合子代与NOD scid小鼠互交；
(c) 选择步骤(b)中Prkdc等位基因纯合并具有更类似于NOD scid的基因型的后代；
(d) 将步骤(c)中选择的后代与Hr等位基因纯合或杂合的小鼠杂交。
10) 条款9的方法，其中所述Prkdc等位基因为scid (Prkdc scid )。
11) 条款9-10中任一项的方法，其中所述Hr等位基因为hr (Hr hr )。
12) 条款9-11中任一项的方法，其中使用以下方法对步骤(a)产生的子代进行基因型分析：
单核苷酸多态性等位基因鉴别(discrimination)、聚合酶链反应和单核苷酸多态性概况分析(profiling)。
13) 条款9-12中任一项的方法，其中步骤(a)和(b)的NOD scid小鼠为NOD.CB17-Prkdc scid 小鼠。
14) 条款9-12中任一项的方法，其中步骤(a)和(b)的NOD scid小鼠为NOD.CB17-Prkdcscid/NCrHsd-Prkdc scid 小鼠。
15) 条款9-14中任一项的方法，其中步骤(a)的第二小鼠品系为MF-1-hr小鼠。
附图简述
图1显示雄性和雌性NOD scid和Hr NOD scid小鼠的肠系膜淋巴结(上部)和脾(下部)的组织切片。
图2显示CD49b的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图3显示CD11c的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图4显示CD3e和CD49b的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图5显示CD3e和CD49b的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图6显示CD3e和CD8α的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图7显示CD49b的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图8显示CD49b的流式细胞术(FACS)结果的点图。象限中的数字为平均百分比。
图9显示从异种移植入Hr NOD scid小鼠中的人套细胞淋巴瘤细胞系JEKO-1的肿瘤发生。
图10显示从异种移植入无胸腺裸鼠中的人套细胞淋巴瘤细胞系JEKO-1的肿瘤发生。
图11显示从异种移植入无全身照射的Hr NOD scid小鼠中的人大细胞淋巴瘤细胞系KARPAS-299的肿瘤发生。
发明详述
尽管本发明可以有各种修饰和替代形式，但本文将详细描述特定实施方案。然而，应理解的是，其无意将本发明限制在所描述的特定形式，恰恰相反，意图是涵盖落入本发明范围的所有修饰、等同物和替代方案。
说明书全文中使用的以下术语意欲具有下列含义：
如本文所使用的，短语“更类似于NOD scid”指在特定的世代中具有比同代其它小鼠更类似NOD scid品系的基因型的小鼠。其可例如，通过单核苷酸多态性分析定量。
本文公开了无毛NOD scid小鼠的生产。无毛NOD scid小鼠对于利用异种移植、自发性肿瘤、癌细胞肿瘤发生、肿瘤血管发生、肿瘤转移潜能、肿瘤抑制治疗、致癌调控、肿瘤成像和人干细胞移入的生物医学研究和开发实验尤其有利。
NOD scid小鼠
Prkdc scid 突变已经从C.B-17背景转移至糖尿病易感的非肥胖糖尿病(NOD)背景，被称为NOD scid。NOD scid突变小鼠由国家癌症研究所(National Cancer Institute)，Frederick，Maryland于2004从国立卫生研究院(National Institutes of Health)，Bethesda，Maryland接收。NOD scid具有影响T细胞和B细胞发育的重度联合免疫缺陷(scid)。与典型NOD小鼠不同的是，自身免疫性糖尿病不因为T细胞功能缺乏而发生。NOD scid小鼠的特征进一步在于具有NK细胞功能缺陷、巨噬细胞缺陷以及无循环补体。NOD scid小鼠对机会致病菌易感，并且具有随年龄增加的胸腺淋巴瘤发病率。
无毛(Hr)小鼠
纯合的(hr/hr)小鼠具有正常的皮毛，直至约10天龄，此时开始脱毛。尽管稀疏的毛生长以每月一次的间隔出现，但小鼠在5-6周龄时基本为无毛的。完整的毛从毛囊消失(Crew和Mirskaia, 1931)。触须重复脱落，并且随年龄增长变得更加异常。无毛基因为甲状腺激素应答基因和甲状腺激素受体的辅阻遏物，介导甲状腺激素在脑和皮肤的作用(Potter等，2001)。这些小鼠的趾甲往往长而弯曲。
一般而言，维持无毛小鼠不需要特殊的饲养程序。除了偶尔的皮肤脓肿，其对感染的易感性通常不增加。然而，无毛小鼠确实有轻度免疫缺陷，可能是由于T-辅助细胞缺陷(Reske-Kunz等，1979)。与hr/+小鼠相比，hr/hr具有高白血病发病率(Heiniger等，1974)并伴随免疫缺陷和受损的移植物抗宿主(GVH)反应(L’Anson和Gasser，1973)。无毛小鼠中缺陷的免疫功能与淋巴瘤发生的关联可由于该突变体对同源淋巴瘤细胞和纯化小鼠白血病病毒(MuLV)的免疫反应降低(Johnson和Meier，1981)。此外，已证明该突变体的脾T细胞对I区同种异体抗原的增殖反应降低(Morrissey等，1980)。此外，已经发现在脾中Ly-1+和Ly-123+ T细胞的正常比例反转(Reske-Kunz等，1979)。
从组织病理学上看，从约14天开始无毛小鼠毛管上部的复层上皮角化过度。杵状毛(Hair club)形成是异常的，且内部根鞘在毛干末端部分周围聚结，使毛鞘下部不能随杵状毛上升而上升，进而滞留在真皮。胞囊从两个源头发育，即角化过度的毛管上部和滞留在真皮的异常毛囊的鞘(Fraser，1946；Mann，1971)。Fukui等，1975描述了无毛小鼠中的肾小球变化。无毛小鼠可生育，但大多数雌性不能很好地哺乳其幼仔。最有效的繁育系统使用纯合的突变体雄性和杂合的雌性(ILAR，1989)。
NK细胞、巨噬细胞和补体功能的存在可影响肿瘤细胞植入后的初期肿瘤生长和转移扩散。各种造血癌细胞系在不同动物模型中的肿瘤生长速率的比较研究显示在B细胞、T细胞、NK细胞、巨噬细胞和补体缺陷的模型中获得较好的生长速率，如在无毛NOD scid小鼠模型中出现的。此外，相对于其它原位小鼠模型，无毛NOD scid小鼠具有高转移潜能。无毛NOD scid小鼠是异种移植和人源化研究的有用模型，并且与其它遗传背景的scid突变体相比具有遗传和表型优势。
在一个实施方案中，公开了非肥胖糖尿病(NOD)背景的无毛、免疫缺陷小鼠。该无毛免疫缺陷小鼠具有B细胞、T细胞、NK细胞、巨噬细胞和补体缺陷。此外，该无毛免疫缺陷小鼠出人意料地具有树突细胞缺陷，其中与有毛的NOD scid小鼠相比，无毛NOD scid小鼠的树突细胞数目减少。树突细胞的减少导致更大程度的免疫缺陷，其对于涉及异种移植、自发性肿瘤、癌细胞肿瘤发生、肿瘤血管发生、肿瘤转移潜能、肿瘤抑制治疗、致癌调控、肿瘤成像和人干细胞移入的实验可为有利的。
实施例
实施例：选择育种以生产无毛NOD scid小鼠
按照表1所示繁育小鼠。概括来说，其步骤如下(突变等位基因的存在用符号+表示)：
步骤(1)：用纯合的NOD scid (Prkdc +/+ )小鼠繁育纯合的无毛(Hr +/+ )小鼠。优选Hr小鼠为雄性，这是因为Hr雌性可显示较差的母性特征。步骤1产生的F1小鼠全部为杂合的：Hr +/- 和Prkdc +/- 。
步骤(2)：选择NOD scid杂合雄性，将其与NOD scid Prkdc +/+ 纯合雌性回交。N2小鼠为基因型的混合。通过PCR检测后代中Hr基因的存在情况，通过单核苷酸多态性等位基因鉴别检测后代中Prkdc基因的存在情况，通过单核苷酸多态性概况分析检测后代中NOD scid遗传背景。
步骤(3)：选择具有至少80.8% NOD scid遗传背景并且Prkdc纯合(Prkdc +/+ )和Hr杂合(Hr +/- )的雄性和雌性，相应地用NOD scid雄性或雌性繁育它们。
步骤(4)：通过PCR检测后代中Hr基因的存在情况，通过单核苷酸多态性概况分析检测后代中NOD scid遗传背景。选择具有至少94% NOD scid遗传背景并且Prkdc纯合(Prkdc +/+ )和Hr杂合(Hr +/- )的雄性和雌性，相应地用NOD scid雄性或雌性繁育它们。
步骤(5)：通过PCR检测后代中Hr基因的存在情况，通过单核苷酸多态性概况分析检测后代中NOD scid遗传背景。选择具有至少99% NOD scid遗传背景并且Prkdc纯合(Prkdc +/+ )和Hr杂合(Hr +/- )的雄性和雌性，相应地用NOD scid雄性或雌性繁育它们。
步骤(6)：通过PCR检测后代中Hr基因的存在情况，通过单核苷酸多态性概况分析检测后代中NOD scid遗传背景。选择具有基本上100% NOD scid遗传背景并且Prkdc纯合(Prkdc +/+ )和Hr杂合(Hr +/- )的雄性和雌性，相应地用NOD scid雄性或雌性繁育它们。
步骤(7)：繁育步骤6得到的小鼠以建立无毛NOD scid群体。Prkdc纯合(Prkdc +/+ )和Hr纯合(Hr +/+ )的雄性可用Prkdc纯合(Prkdc +/+ )和Hr纯合(Hr +/+ )或杂合(Hr +/- )的雌性繁育。
表1：生产无毛NOD scid小鼠的繁育图解
可任选使用具有导致无毛表型的除Hr hr 外无毛基因自发或定向突变的小鼠，包括但不限于Hr rh 、Hr ba 、Hr n 、hr rh8j 、hr rhChr 、hr rhsl 、hr rhy 、hr bmh 和hr TgN5053Mm 。
无毛NOD scid小鼠的表型
定时对无毛NOD scid小鼠称重用于生长曲线分析。此外，小鼠接受标准化学板(standard chemistry panel)、全血测试和骨髓涂片。使用流式细胞术检测B细胞和T细胞标志、NK细胞、巨噬细胞和补体的存在情况。进一步的测试包括胸腺、脾脏、骨髓和淋巴结以及皮肤(乳房脂肪垫和真皮)的组织病理学。
还实施本领域技术人员已知的测试以测定无毛NOD scid小鼠的免疫缺陷，包括检测免疫球蛋白水平以及NK细胞、巨噬细胞和补体活性。还监测小鼠自发性淋巴瘤的发病率。
表2：无毛NOD scid小鼠与BALB/c和NOD scid的全血计数和免疫球蛋白水平比较
| 小鼠 | 淋巴细胞/μl | IgG(MFI计数) | IgG(ng/μl，估算) |
| Hr NOD scid雄性 | 286 | 4622 | 2-5 |
| Hr NOD scid雌性 | 528 | 7563 | 2-5 |
| BALB/c雄性 | 5308 | 16252 | 80 |
| BALB/c雌性 | 8670 | 17575 | 80 |
| NOD scid雄性 | 1857 | 7883 | 2-5 |
| NOD scid雌性 | 1809 | 6109 | 2-5 |
实施例：Hr NOD scid与NOD scid小鼠的全血计数比较
对动物实施安乐死，并末端取血用于CBC和血清抗体(Ab)测量。实施常规CBC。白细胞计数、红细胞和血小板参数在HEMAVET 950FS自动血液学分析仪上测量。白细胞分类(differential)得自取血时获得的血液涂片。使用该手动分类结果计算各白细胞类型的估计数目。
表3：无毛NOD scid与NOD scid小鼠的全血计数比较
组织病理学
比较Hr NOD scid和NOD scid小鼠之间的组织病理学指数。收集腋窝、腹股沟、颈部/下颌骨和肠系膜淋巴结以及脾脏、胸腺、乳房组织、盲肠和胰腺。骨髓涂片从股骨髓收集，并收集胸骨和股骨用于组织学分析。
在无毛NOD scid小鼠中，淋巴结和脾白髓无具有典型淋巴细胞形态的细胞(稠核、胞质稀少)。残留的可见细胞为网状支持细胞、树突细胞、巨噬细胞和NK细胞。在脾红髓中有不同程度的血细胞生成。
免疫细胞标志
实施例：免疫球蛋白。通过多重荧光免疫测定(MFI)测量血清抗体。Hr NOD scid小鼠的所有IgG测量值均小于6.25μg/mL，或在SCID模型的渗漏阈值以下。
实施例：脾细胞表面抗原的荧光活化细胞分选术(FACS)。分析细胞表面抗原以测定Hr NOD scid小鼠与NOD scid小鼠相比的免疫细胞缺陷类型和水平。
材料和方法
脾细胞收集。将新鲜去除的脾脏置于含有2 ml完全RPMI-10 (450 ml RPMI-1640，50Ml FBS，7.5 ml 1M HEPES，pH7.2)的培养皿中。用解剖刀片将脾脏切成多个小块。用5 ml注射器的活塞采用圆周运动将脾脏碎片压向培养皿底部，直至大部分纤维组织残留。通过用20 g针将混悬液吸进并排出5 ml注射器多次使团块进一步分散在混悬液中。通过细胞滤器，将混悬液排出至Falcon 12 x 75 mm聚丙烯管中。
除去红细胞。向脾细胞混悬液中加入两毫升PHARM LYSE (用Milli-Q水1:10稀释)。然后立即将混悬液涡旋，并在黑暗中室温孵育15分钟。然后将混悬液于4℃ 200 x g离心5分钟。在不扰动沉淀的情况下，小心将上清液倒出，用纸巾吸干试管的边缘。接着将沉淀重悬在1 ml冷的染色缓冲液中。使混悬液通过细胞滤器至干净FALCON 12 x 75 mm聚丙烯管(其保持在冰上)中。
细胞计数。将50微升台盼蓝(Trypan Blue)、90 μl染色缓冲液和10 μl细胞混悬液加入至0.5 ml eppendorf管中。彻底混合后，将30 μl加在血细胞计数器上。在10X物镜中对活细胞进行计数(活细胞出现折射，而死细胞为蓝色)。接着将沉淀在350 x g离心5分钟，随后重悬至合适的终体积(用于FACS分析的每200 μl样品中1 x 106个细胞) 并等分至1.5 ml管中。
细胞表面抗原的荧光免疫染色。用荧光染料缀合的细胞表面抗原特异性单克隆抗体对细胞进行免疫染色。用抗体在黑暗中4℃孵育细胞30分钟。然后通过500 x 5离心5分钟使其沉淀，接着用500 μl冷的染色缓冲液重悬，并转移至FALCON 12 x 75 mm聚苯乙烯中。除去可见团块，然后进行FACS分析。
表4：抗体如下
结果
流式细胞术分析证实Hr NOD scid小鼠几乎完全缺乏T细胞和B细胞。CD19(常见的B细胞谱系标志)显示两品系均几乎完全没有成熟B细胞(图5)。Hr NOD scid小鼠的淋巴细胞总数比NOD scid小鼠少。特别地，与NOD scid相比，Hr NOD scid小鼠中的NK细胞(图2)和树突细胞(图3)显著减少。两品系的T细胞和NK细胞亚群以及树突细胞存在统计上的显著差异。使用SAS对流式细胞术数据进行双向ANOVA分析。0.05或更小的P值指示显著性差异(Hr NOD scid，n = 12；NOD scid，n = 10)。
使用下表中细胞表面抗原的正染色区别不同的类型的免疫细胞。
表5
| 细胞表面抗原 | 细胞类型 |
| CD3e | T细胞 |
| CD4 | 辅助性T细胞 |
| CD8α | 细胞毒性T细胞 |
| CD11c | 树突细胞 |
| CD19 | B细胞 |
| CD49b | NK细胞 |
实施例：致瘤细胞系的异种移植
依照本领域已知的程序将衍生自膀胱癌、乳腺癌、头颈癌、结肠癌、血癌、肝癌、肺癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌或睾丸癌的致瘤细胞系移植入无毛NOD scid小鼠中。检测下列细胞系在无毛NOD scid小鼠中被摄取和生长的能力：HTLA-230 (Brignole等，2004)、CT-26 (Guba等，2001)、BW5147 (Geldhof等，1998)、Grant 519 (Wang等，2007)、Karpas 299 (Francisco等，2003)、L540cy (Francisco等，2003)、Colo-699 (Boehle等，2001)、SIT1 (Bromberg等，2005)、TAC 7 (Bromberg等，2005)、DU-145 (Glinskii等，2005)。
人大细胞淋巴瘤细胞系KARPAS-299和套细胞淋巴瘤细胞系JEKO-1购买自ATCC。KARPAS-299细胞在含有10% FBS的RPMI 1640中培养，JEKO-1在含有20% FBS的RPMI 1640中培养。用与细胞混悬液1:1稀释的基质胶(Matrigel)，用1.0 X 107个细胞皮下接种约八周龄的雌性小鼠。
KARPAS-299细胞系在没有全身照射的无胸腺小鼠中将不生长。与此相反，Hr NOD scid模型在无照射下皮下注射后表现出强劲的生长(图11)。JEKO-1肿瘤细胞系可成功移植入无胸腺裸鼠，无论其在接种前是否照射，但初期增殖缓慢。在本研究中，无胸腺裸鼠的对数期生长具有约25-27天的时滞(图9)，而Hr NOD scid小鼠显示快速和有力生长(图10)。实际上，所有的Hr NOD scid小鼠在接种后14天内出现肿瘤。
测试了额外细胞系在无毛NOD scid小鼠中的摄取和生长情况，所述额外细胞系包括：U251-NG、MCF-7、MDA-MB-231、T47D、CX-1、DLD-1、HAC-7、HC116、HT-29、FaDu、HNX-OE、A549、H322、H358、LL2、NCI-8460、NCI-N417、B16F10、MOT、OVCAR3、CFPAC-1、Colo57、MlaPaca-2、PANC-1、PL45、CWR22、LNAI、PC-3、NCCIT、T24、G55、U87MG、SH-SY5Y、MCF-7、MT-1、HPB-ALL、HepT1、Hep3B、HuH6和SKOV3。还测试了其它细胞类型例如干细胞(表达CD34)植入时健壮生长的能力。
出于比较目的，实验中可包括其它小鼠品系，例如NOD scid、无胸腺裸鼠、MF-1无毛、CB.17、BALB/c等。
使用单核苷酸多态性(SNP)标记-辅助的回交方法，已将无毛突变(Hr hr )从远交的HsdOla:MF1-Hr hr 转移至NOD.CB17-Prkdc scid /NCrHsd小鼠，以产生近交无毛NOD.Cg-Prkdc scid Hr hr /NCrHsd小鼠，通常称为无毛NOD scid。本文描述了该模型的免疫学特征，其包括B细胞和T细胞缺陷、NK细胞功能缺陷以及巨噬细胞和补体功能降低。此外，本文发现与NOD scid相比Hr NOD scid小鼠中NK细胞和树突细胞大大减少。
尽管前面的说明书已经对本发明进行详细说明和描述，但这些说明和描述在性质上应被视为示例，而非限制性的，应理解的是，仅描述了说明性实施方案，但落入本发明范围内的所有变化和修饰均要求受到保护。本领域普通技术人员可容易地结合本文描述的一个或多个特征设计其自己的实现方案，因此落入本发明范围内。
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Claims (13)
1. 非肥胖糖尿病(NOD)背景下的无毛、免疫缺陷小鼠，其具有B细胞、T细胞、NK细胞、巨噬细胞和补体缺陷。
2. 权利要求1的小鼠，其中所述小鼠为Prkdc等位基因纯合的。
3. 权利要求2的小鼠，其中所述Prkdc等位基因为scid (Prkdc scid )。
4. 权利要求1-3中任一项的小鼠，其中所述小鼠为Hr等位基因纯合的。
5. 权利要求4的小鼠，其中所述Hr等位基因为hr (Hr hr )。
6. 权利要求1-5中任一项的小鼠，其中所述非肥胖糖尿病背景为NOD.CB17-Prkdc scid 。
7. 权利要求1-6中任一项的无毛小鼠，进一步具有树突细胞缺陷，其中所述树突细胞缺陷为与相同背景而非无毛的免疫缺陷小鼠相比树突细胞的数目减少。
8. 生产非肥胖糖尿病(NOD)背景下的无毛、免疫缺陷小鼠的方法，所述方法包括：
(a) 将Prkdc等位基因纯合的NOD scid小鼠品系与Hr等位基因纯合的第二小鼠品系杂交，以产生Hr等位基因和Prkdc等位基因均杂合的子代；
(b) 将步骤(a)产生的杂合子代与NOD scid小鼠互交；
(c) 选择步骤(b)中Prkdc等位基因纯合并具有更类似于NOD scid的基因型的后代；
(d) 将步骤(c)中选择的后代与Hr等位基因纯合或杂合的小鼠杂交。
9. 权利要求8的方法，其中所述Prkdc等位基因为scid (Prkdc scid )。
10. 权利要求8-9中任一项的方法，其中所述Hr等位基因为hr (Hr hr )。
11. 权利要求8-10中任一项的方法，其中使用选自以下的方法对步骤(a)产生的子代进行基因型分析：
单核苷酸多态性等位基因鉴别、聚合酶链反应和单核苷酸多态性概况分析。
12. 权利要求8-11中任一项的方法，其中步骤(a)和(b)的NOD scid小鼠为NOD.CB17-Prkdc scid 小鼠。
13. 权利要求8-12中任一项的方法，其中步骤(a)的第二小鼠品系为MF-1-hr小鼠。
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| CN110868850A (zh) * | 2017-05-12 | 2020-03-06 | 杰克逊实验室 | 缺乏i类和ii类mhc的nsg小鼠 |
| CN111838066A (zh) * | 2019-07-12 | 2020-10-30 | 上海吉辉实验动物饲养有限公司 | 一种自发性突变无毛SHJHhr/hr早衰老小鼠动物模型及其培育方法 |
| US11712026B2 (en) | 2017-10-18 | 2023-08-01 | The Jackson Laboratory | Murine-MHC-deficient HLA-transgenic nod-mouse models for T1D therapy development |
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| US20090235372A1 (en) * | 2008-03-13 | 2009-09-17 | Clifford Charles B | Hairless Immunodeficient Mouse Model |
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| WO2005090968A1 (en) * | 2004-03-16 | 2005-09-29 | Inist Inc. | Tat-based immunomodulatory compositions and methods of their discovery and use |
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- 2011-04-14 EP EP11769588.2A patent/EP2558576B1/en not_active Not-in-force
- 2011-04-14 US US13/640,522 patent/US9018441B2/en not_active Expired - Fee Related
- 2011-04-14 CN CN201180029157.1A patent/CN102971420B/zh not_active Expired - Fee Related
- 2011-04-14 ES ES11769588.2T patent/ES2601800T3/es active Active
Patent Citations (1)
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| US20090235372A1 (en) * | 2008-03-13 | 2009-09-17 | Clifford Charles B | Hairless Immunodeficient Mouse Model |
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| DALE L. GREINER ET AL: "SCID Mouse Models of Human Stem Cell Engraftment", 《 STEM CELLS》 * |
| 宋艳秋等: "K652/NOD-SCID小鼠白血病模型的建立", 《中国实验血液学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110868850A (zh) * | 2017-05-12 | 2020-03-06 | 杰克逊实验室 | 缺乏i类和ii类mhc的nsg小鼠 |
| US11778994B2 (en) | 2017-05-12 | 2023-10-10 | The Jackson Laboratory | NSG mice lacking MHC class I and class II |
| US11712026B2 (en) | 2017-10-18 | 2023-08-01 | The Jackson Laboratory | Murine-MHC-deficient HLA-transgenic nod-mouse models for T1D therapy development |
| CN111838066A (zh) * | 2019-07-12 | 2020-10-30 | 上海吉辉实验动物饲养有限公司 | 一种自发性突变无毛SHJHhr/hr早衰老小鼠动物模型及其培育方法 |
| CN111838066B (zh) * | 2019-07-12 | 2022-10-14 | 上海吉辉实验动物饲养有限公司 | 一种自发性突变无毛SHJHhr/hr早衰老小鼠动物模型培育方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2601800T3 (es) | 2017-02-16 |
| EP2558576A1 (en) | 2013-02-20 |
| EP2558576B1 (en) | 2016-10-19 |
| US9018441B2 (en) | 2015-04-28 |
| CN102971420B (zh) | 2016-10-26 |
| WO2011130512A1 (en) | 2011-10-20 |
| EP2558576A4 (en) | 2013-12-04 |
| US20130205417A1 (en) | 2013-08-08 |
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