CN110402892A - 选择性敲除胰腺上皮细胞程序性死亡配体1分子的自发胰腺癌小鼠模型的建立方法 - Google Patents
选择性敲除胰腺上皮细胞程序性死亡配体1分子的自发胰腺癌小鼠模型的建立方法 Download PDFInfo
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Abstract
本发明公开了一种选择性敲除胰腺上皮细胞程序性死亡配体1分子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选择性敲除PD‑L1基因同时表达持续活化的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，从而获得肿瘤细胞PD‑L1分子表达阴性的自发胰腺癌小鼠模型。本发明的有益效果为：本发明构建的胰腺上皮组织PD‑L1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，且胰腺上皮组织来源的癌细胞中不表达PD‑L1分子，该模型可用于研究胰腺上皮细胞PD‑L1分子表达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，主要是一种一种选择性敲除胰腺上皮细胞程序性死亡配体1分子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，我们设计并构建了PD-L1flox/flox小鼠并通过杂交手段将其整合入KTC小鼠得到胰腺癌细胞特异性敲除PD-L1的基因工程小鼠模型。该小鼠模型对肿瘤来源PD-L1影响胰腺癌的生物学行为、免疫状态改变等研究以及相关药物研发都具有重要意义。
发明内容
本发明的目的在于克服现有技术存在的不足，而提供一种选择性敲除胰腺上皮细胞程序性死亡配体1(PD-L1)分子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。一种选择性敲除胰腺上皮细胞程序性死亡配体1分子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选择性敲除PD-L1基因同时表达持续活化的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，从而获得肿瘤细胞PD-L1分子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)、自行构建了PD-L1flox/flox转基因小鼠；自美国Jackson Lab购得KrasLSL-G12D/+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；
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420b；
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:CAS099-LOXP-F1:5’-ACA TCC AGA CCT TAG AGT AAC CG-3’；CAS099-LOXP-R1:5’-TAC ATT CAA ACT CAG CCA AGG AC-3’；LoxP产物条带为366bp，野生型产物条带为293bp；
筛选得到KrasLSL-G12D/+TGF-βR2flox/floxPD-L1flox/floxPtf1a-Cre小鼠，同时具有Kras突变条带、TGF-βR2LoxP条带、PD-L1LoxP条带、Ptf1a-Cre条带的即为目的KTC-PLoxP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30ulpH6.8TrisHCL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。
本发明的有益效果为：本发明构建的胰腺上皮组织PD-L1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，且胰腺上皮组织来源的癌细胞中不表达PD-L1分子，该模型可用于研究胰腺上皮细胞PD-L1分子表达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是PD-L1条件性敲除小鼠基因组LoxP位点插入示意图；
图2是LoxP位点小鼠基因组测序验证。
图3为KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1Flox/FloxPtf1a-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：
一种选择性敲除胰腺上皮细胞程序性死亡配体1分子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(ptf1a)驱动的Cre-loxp系统，在小鼠胰腺上皮组织中选择性敲除PD-L1基因同时表达持续活化的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，从而获得肿瘤细胞PD-L1分子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)自行构建了PD-L1flox/flox基因工程小鼠；自美国Jackson Lab购得KrasLSL-G12D/+基因工程小鼠；经美国Vanderbilt-Ingram癌症中心Harold Moses教授处获赠TGF-βR2flox /flox，Ptf1a-Cre基因工程小鼠。
2)小鼠杂交:将不同基因型小鼠建立Breeder，并进行杂交。
3)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30ulpH6.8TrisHCL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。
4)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:CAS099-LOXP-F1:5’-ACA TCC AGA CCT TAG AGT AAC CG-3’；CAS099-LOXP-R1:5’-TAC ATT CAA ACT CAG CCA AGG AC-3’；LoxP产物条带为366bp，野生型产物条带为293bp。
5)同时具有Kras突变条带、TGF-βR2LoxP条带(纯合子或杂合子)、PD-L1LoxP条带(纯合子或杂合子)、Ptf1a-Cre条带的即为目的KTC-PLoxP小鼠。
图1是PD-L1条件性敲除小鼠基因组LoxP位点插入示意图；图2是LoxP位点小鼠基因组测序验证，图3为KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1Flox/FloxPtf1a-Cre小鼠基因型鉴定，红框所标记的#5为阳性目的小鼠；KTC-PLoxP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-L1Flox/+,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-L1Flox/+Ptf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/+PD-L1Flox/FloxPtf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1Flox/+Ptf1a-Cre、
KrasLSL-G12D/+TGF-βR2Flox/FloxPD-L1Flox/FloxPtf1a-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)
1.一种选择性敲除胰腺上皮细胞程序性死亡配体1分子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选择性敲除PD-L1基因同时表达持续活化的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，从而获得肿瘤细胞PD-L1分子表达阴性的自发胰腺癌小鼠模型。
2.根据权利要求1所述的选择性敲除胰腺上皮细胞程序性死亡配体1分子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)、自行构建了PD-L1flox/flox转基因小鼠；自美国Jackson Lab购得KrasLSL-G12D/+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；
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420b；
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:CAS099-LOXP-F1:5’-ACA TCC AGA CCT TAG AGT AAC CG-3’；CAS099-LOXP-R1:5’-TAC ATT CAA ACT CAG CCA AGG AC-3’；LoxP产物条带为366bp，野生型产物条带为293bp；
筛选得到KrasLSL-G12D/+TGF-βR2flox/floxPD-L1flox/floxPtf1a-Cre小鼠，同时具有Kras突变条带、TGF-βR2LoxP条带、PD-L1 LoxP条带、Ptf1a-Cre条带的即为目的KTC-PLoxP小鼠。
3.根据权利要求2所述的选择性敲除胰腺上皮细胞程序性死亡配体1分子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。
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