CN110408654A - 程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法 - Google Patents
程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法 Download PDFInfo
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Abstract
本发明公开了一种程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，该方法包括如下步骤：通过胰腺转录因子1a驱动的Cre‑loxp系统，在小鼠胰腺上皮组织中表达持续活化的K‑rasG12D突变，并选择性敲除TGF‑βR2基因，使小鼠在短期内自发形成胰腺癌，同时敲除所有组织细胞中PD‑L1分子，从而获得肿瘤细胞与间质细胞PD‑L1分子均表达阴性的自发胰腺癌小鼠模型。本发明的有益效果为：本发明构建的PD‑L1完全敲除的自发胰腺癌小鼠模型，其特点为小鼠胰腺组织可自发形成胰腺癌，且胰腺癌细胞与所有间质细胞均不表达PD‑L1分子，该模型可用于研究PD‑L1分子在小鼠胰腺癌发生发展过程，尤其是免疫逃逸机制的活体研究。
Description
技术领域
本发明涉及一种基因工程小鼠模型的构建方法，主要是一种程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法。
背景技术
胰腺导管腺癌(Pancreatic ductal adenocarcinoma,PDAC)是最恶性的胰腺肿瘤，确诊后五年生存率往往不足5％，被称为“癌中之王”[1,2]。KrasLSL-G12D/+；TGF-βR2flox/flox；Ptf1a-Cre(KTC)小鼠可以自然进展为侵袭性胰腺癌，且忠实还原人类胰腺癌的临床及组织学特征[3]。随着全球抗肿瘤免疫治疗的浪潮兴起，以抗PD-L1/PD-1为代表的免疫检查点抑制治疗相继涌现，而该疗法目前在胰腺癌中未得到确切疗效[4]。为研究不同组织细胞来源的PD-L1在胰腺癌中的作用方式以及胰腺癌对该疗法的抵抗或代偿机制，我们将PD-L1-/-小鼠通过杂交手段将其基因型引入KTC小鼠得到全身敲除PD-L1的自发胰腺癌基因工程小鼠模型。该小鼠模型有助于研究多种来源的PD-L1分子对胰腺癌的生物学行为、免疫状态的影响，对药物研发也具有重要意义。
发明内容
本发明的目的在于克服现有技术存在的不足，而提供一种程序性死亡配体1(PD-L1)分子完全敲除的自发胰腺癌小鼠模型的建立方法。
本发明的目的是通过如下技术方案来完成的。一种程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，该方法包括如下步骤：通过胰腺转录因子1a驱动的Cre-loxp系统，在小鼠胰腺上皮组织中表达持续活化的K-rasG12D突变，并选择性敲除TGF-βR2基因，使小鼠在短期内自发形成胰腺癌，同时敲除所有组织细胞中PD-L1分子，从而获得肿瘤细胞与间质细胞PD-L1分子均表达阴性的自发胰腺癌小鼠模型。
更进一步的，该方法包括如下步骤：
(1)、自美国Jackson Lab购得KrasLSL-G12D/+、PD-L1+/-转基因小鼠；经美国Vanderbilt-Ingram癌症中心Harold Moses教授处获赠TGF-βR2flox/flox，Ptf1a-cre转基因小鼠；
(2)、将上述四种小鼠进行杂交繁殖；
(3)、构建引物，对杂交的小鼠后代进行基因型鉴定，筛选得到KrasLSL-G12D/+TGF-βR2flox/floxPD-L1-/-Ptf1a-Cre小鼠，即KTC-PKO小鼠。
更进一步的，步骤(3)中子代小鼠分别剪取鼠尾或脚趾，放入EP管中，加入300ul50mMNaOH，金属浴100℃煮1小时，加入30ulpH6.8 TrisHCL中和；12000转/分离心5分钟，上清即为制备好的鼠尾DNA模版液，用于进行后续的PCR鉴定；同时具有Kras突变条带、TGF-βR2LoxP条带、PD-L1KO条带、Ptf1a-Cre条带的即为目的KTC-PKO小鼠。
本发明的有益效果为：本发明构建的PD-L1完全敲除的自发胰腺癌小鼠模型，其特点为小鼠胰腺组织可自发形成胰腺癌，且胰腺癌细胞与所有间质细胞均不表达PD-L1分子，该模型可用于研究PD-L1分子在小鼠胰腺癌发生发展过程，尤其是免疫逃逸机制的活体研究。
附图说明
图1为KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1-/-Ptf1a-Cre小鼠基因型鉴定示意图。
具体实施方式
下面将结合附图对本发明做详细的介绍：
一种程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，通过胰腺转录因子1a驱动的Cre-loxp系统，在小鼠胰腺上皮组织中表达持续活化的K-rasG12D突变，并选择性敲除TGF-βR2基因，使小鼠在短期内自发形成胰腺癌，同时敲除所有组织细胞中PD-L1分子(PD-L1-/-)，从而获得肿瘤细胞与间质细胞PD-L1分子均表达阴性的自发胰腺癌小鼠模型。
该方法具体包括如下步骤：
(1)、自美国Jackson Lab购得KrasLSL-G12D/+、PD-L1+/-转基因小鼠；经美国Vanderbilt-Ingram癌症中心Harold Moses教授处获赠TGF-βR2flox/flox，Ptf1a-cre转基因小鼠；
(2)、将上述四种小鼠进行杂交繁殖；
(3)、构建引物，对杂交的小鼠后代进行基因型鉴定，筛选得到KrasLSL-G12D/+TGF-βR2flox/floxPD-L1-/-Ptf1a-Cre小鼠，即KTC-PKO小鼠。
具体为：子代小鼠分别剪取鼠尾或脚趾，放入EP管中，加入300ul 50mMNaOH，金属浴100℃煮1小时，加入30ulpH6.8 TrisHCL中和；12000转/分离心5分钟，上清即为制备好的鼠尾DNA模版液，用于进行后续的PCR鉴定；
PCR鉴定小鼠的基因型：
引物序列：
TGF-βR2:T004:5’-TAAACA AGGTCCGGAGCCCA-3’,T005:ATATCTGCAAGAGGTCCCCT；LoxP产物条带为540bp，野生型产物条带为420bp。
Kras:oIMR9592:5’-GCAGGTCGAGGGACCTAATA-3’,22908:5’-CTGCATAGTACGCTATACCC GT-3’；突变产物条带为100bp。
Ptf1a-Cre:Cre1:5’-ATA GGCTACCTGGCCATGCCC-3’；Cre2:5’-CGG GCT GCAGGAATTCGT CG-3’；突变产物条带为210bp。
PD-L1:10685:5’-CTAACAGGTGATCCGTTTCCTATG-3’；10686:5’-GCCGTGATAGTAAACGCTGAA-3’；野生型产物条带约为330bp；oIMR6046:5’-ATT GAA CAA GATGGA TTG CAC-3’；oIMR6047:5’-CGTCCAGATCATCCTGATC-3’；突变型产物条带约为500bp。
同时具有Kras突变条带、TGF-βR2LoxP条带(纯合子或杂合子)、PD-L1KO条带(纯合子或杂合子)、Ptf1a-Cre条带的即为目的KTC-PKO小鼠。
图1为KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1-/-Ptf1a-Cre小鼠基因型鉴定示意图，红框所标记的#7为阳性目的小鼠，KTC-PKO小鼠仍可自发形成胰腺癌。
本专利所适用的范围基于KrasLSL-G12D/+,TGF-βR2Flox/+,PD-L1-/-,Ptf1a-Cre四种基因型小鼠杂交所产生的后代，包括但不限定于:
KrasLSL-G12D/+TGF-βR2Flox/+PD-L1+/-Ptf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/+PD-L1-/-Ptf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1+/-Ptf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1-/-Ptf1a-Cre。
可以理解的是，对本领域技术人员来说，对本发明的技术方案及发明构思加以等同替换或改变都应属于本发明所附的权利要求的保护范围。
Claims (3)
1.一种程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，其特征在于：该方法包括如下步骤：通过胰腺转录因子1a驱动的Cre-loxp系统，在小鼠胰腺上皮组织中表达持续活化的K-rasG12D突变，并选择性敲除TGF-βR2基因，使小鼠在短期内自发形成胰腺癌，同时敲除所有组织细胞中PD-L1分子，从而获得肿瘤细胞与间质细胞PD-L1分子均表达阴性的自发胰腺癌小鼠模型。
2.根据权利要求1所述的程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，其特征在于：该方法包括如下步骤：
(1)、自美国Jackson Lab购得KrasLSL-G12D/+、PD-L1+/-转基因小鼠；经美国Vanderbilt-Ingram癌症中心Harold Moses教授处获赠TGF-βR2flox/flox，Ptf1a-cre转基因小鼠；
(2)、将上述四种小鼠进行杂交繁殖；
(3)、构建引物，对杂交的小鼠后代进行基因型鉴定；
PCR鉴定小鼠的基因型，引物序列：
TGF-βR2:T004:5’-TAAACA AGGTCCGGAGCCCA-3’,T005:ATATCTGCAAGAGGTCCCCT；LoxP产物条带为540bp，野生型产物条带为420bp；
Kras:oIMR9592:5’-GCAGGTCGAGGGACCTAATA-3’,22908:5’-CTGCATAGTACGCTATACCCGT-3’；突变产物条带为100bp；
Ptf1a-Cre:Cre1:5’-ATA GGCTACCTGGCCATGCCC-3’；Cre2:5’-CGG GCT GCAGGA ATTCGTCG-3’；突变产物条带为210bp；
PD-L1:10685:5’-CTAACAGGTGATCCGTTTCCTATG-3’；10686:5’-GCCGTGATAGTAAACGCTGAA-3’；野生型产物条带约为330bp；oIMR6046:5’-ATT GAA CAA GATGGA TTG CAC-3’；oIMR6047:5’-CGTCCAGATCATCCTGATC-3’；突变型产物条带约为500bp；
筛选得到KrasLSL-G12D/+TGF-βR2flox/floxPD-L1-/-Ptf1a-Cre小鼠，同时具有Kras突变条带、TGF-βR2LoxP条带、PD-L1 KO条带、Ptf1a-Cre条带的即为目的KTC-PKO小鼠。
3.根据权利要求2所述的程序性死亡配体1分子完全敲除的自发胰腺癌小鼠模型的建立方法，其特征在于：步骤(3)中子代小鼠分别剪取鼠尾或脚趾，放入EP管中，加入300ul50mMNaOH，金属浴100℃煮1小时，加入30ulpH6.8 TrisHCL中和；12000转/分离心5分钟，上清即为制备好的鼠尾DNA模版液，用于进行后续的PCR鉴定。
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