CN117402879B - 一种suv39h1基因条件性敲除小鼠的构建方法 - Google Patents
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Abstract
本发明公开了一种SUV39H1基因条件性敲除小鼠的构建方法，涉及动物模型构建技术领域。本发明提供靶向小鼠SUV39H1基因的sgRNA，所述sgRNA包括5’Guide序列和3’Guide序列；所述5’Guide序列包括SEQ ID NO.1～SEQ ID NO.7任一条所示的序列；所述3’Guide序列包括SEQ ID NO.8～SEQ ID NO.14任一条所示的序列。本发明基于CRISPR/Cas9技术，通过所述sgRNA并经过筛选和杂交，构建得到SUV39H1基因条件性敲除小鼠。本发明的SUV39H1基因条件性敲除小鼠的构建方法高效、快捷、简便，得到的SUV39H1基因条件性敲除小鼠稳定性好，为研究SUV39H1基因的功能和在不同生理和病理情境下的作用提供经济、可靠的动物模型。
Description
技术领域
本发明涉及动物模型构建技术领域，具体涉及一种SUV39H1基因条件性敲除小鼠的构建方法。
背景技术
当谈到转基因鼠的构建背景时，CRISPR-Cas9(Clustered RegularlyInterspaced Short Palindromic Repeats Cas9)技术无疑是一个令人兴奋和革命性的工具。这项技术源自细菌和古细菌在长期演化过程中形成的一种适应性免疫防御机制，用于对抗入侵的病毒和外源DNA。它是基因编辑领域的一颗璀璨明珠，被誉为基因工程的"剪刀"。CRISPR-Cas9已经成为基因编辑技术中的第三代，取代了之前ZFN和TALENs技术。它之所以备受欢迎，原因在于它的高效性、简便性、低成本以及容易上手。这项技术使科学家能够准确地编辑生物体的基因，开辟了前所未有的研究和治疗途径。
在CRISPR-Cas9基因编辑技术中，关键的元素之一是sgRNA(引导RNA)。sgRNA是人工设计的RNA分子，它的作用是识别并定位到目标基因组中的特定序列。一旦sgRNA与目标序列匹配，它将引导Cas9蛋白酶精确地切割DNA双链，造成双链断裂。这一断裂可以通过细胞的自然修复机制进行修复，包括非同源末端连接(NHEJ)和同源重组定向修复(HDR)。这种修复过程允许科学家们修改基因组，以实现各种研究和治疗目的。值得注意的是，sgRNA的目标识别能力取决于其具体的序列。通常情况下，当基因之间的间隔较大时，sgRNA对靶序列的错误识别不会导致严重后果。然而，当基因位于目标位置上下游较近的情况下，可能会干扰其他基因的结构，这可能会对转基因结果产生不利影响。
总之，CRISPR-Cas9技术为转基因鼠的构建提供了前所未有的工具和机会，为科学家们在基因研究和治疗方面开辟了广阔的前景。
发明内容
本发明的目的在于克服现有技术的不足，提供一种SUV39H1基因条件性敲除小鼠的构建方法。
为实现上述目的，本发明采取的技术方案为：一种靶向小鼠SUV39H1基因的sgRNA，所述sgRNA包括5’Guide序列和3’Guide序列；所述5’Guide序列包括SEQ ID NO.1～SEQ IDNO.7任一条所示的序列；所述3’Guide序列包括SEQ ID NO.8～SEQ ID NO.14任一条所示的序列。
SUV39H1基因在细胞和生物学研究中具有至关重要的作用，是一种编码组蛋白转移酶的基因，通过催化组蛋白修饰，调节着色体的结构和功能。SUV39H1基因的功能影响着细胞的正常分化、发育和细胞周期等关键生物学过程。此外，SUV39H1异常表达与多种疾病的发生和发展密切相关。本发明针对SUV39H1基因的exon2-3区域设计了sgRNAs，这些区域在SUV39H1功能中起关键作用。本发明将sgRNAs分别放置在intron1和intron3的非保守区域，以确保精确的靶向。本发明的靶向小鼠SUV39H1基因的sgRNA能够在特定条件下选择性地局部或者全身敲除SUV39H1的exon2-3区域，从而研究其在基因表达和染色体结构调控中的确切作用。
作为本发明所述靶向小鼠SUV39H1基因的sgRNA的优选实施方式，所述5’Guide序列如SEQ ID NO.4所示；所述3’Guide序列如SEQ ID NO.10所示。
作为本发明所述靶向小鼠SUV39H1基因的sgRNA的优选实施方式，所述sgRNA的识别位点为SUV39H1基因的第2～3号外显子的两侧。
本发明还提供一种SUV39H1基因条件性敲除小鼠的构建方法，包括以下步骤：
(1)设计筛选靶向小鼠SUV39H1基因的sgRNA；
(2)将sgRNA、Cas9蛋白和打靶载体通过显微注射到小鼠受精卵中，并将该受精卵移植至假孕小鼠，对假孕小鼠所生的仔鼠进行基因型鉴定，得F0代小鼠；
(3)将F0代小鼠与野生型小鼠交配，得F1代小鼠；
(4)经基因型鉴定筛选F1代杂合子小鼠与Cre工具鼠交配，得所述SUV39H1基因条件性敲除小鼠。
作为本发明所述SUV39H1基因条件性敲除小鼠的构建方法的优选实施方式，所述步骤(1)中的sgRNA的核苷酸序列如SEQ ID NO.4和SEQ ID NO.10所示。
作为本发明所述SUV39H1基因条件性敲除小鼠的构建方法的优选实施方式，所述F0代和F1代小鼠的基因型鉴定使用的5’端鉴定引物如SEQ ID NO.15和SEQ ID NO.16所示，3’端鉴定引物如SEQ ID NO.17和SEQ ID NO.18所示。
作为本发明所述SUV39H1基因条件性敲除小鼠的构建方法的优选实施方式，所述F0代和F1代小鼠的基因型鉴定时，PCR扩增的程序为94℃预变性5min；98℃变性30s，67℃退火30s，68℃延伸1kb/min，每个循环退火温度降低0.7℃，共15个循环；98℃变性30s，57℃退火30s，68℃延伸1kb/min，25个循环；68℃延伸10min。
本发明还提供所述的SUV39H1基因条件性敲除小鼠的构建方法构建得到的SUV39H1基因条件性敲除小鼠。
本发明还提供所述SUV39H1基因条件性敲除小鼠在研究SUV39H1基因相关功能和作用机制中的应用。通过比较SUV39H1基因条件性敲除小鼠和野生型小鼠的生存率和健康状况，可研究SUV39H1在整体健康中的作用；通过研究条件性敲除小鼠的生殖能力，可了解SUV39H1对生育和生殖的影响。
本发明还提供所述SUV39H1基因条件性敲除小鼠在筛选或制备用于治疗与SUV39H1基因相关疾病的药物中的应用。
本发明的有益效果：本发明提供一种靶向小鼠SUV39H1基因的sgRNA，所述sgRNA靶向识别SUV39H1基因第2～3号外显子的两侧。基于CRISPR/Cas9技术，通过所述sgRNA并经过筛选和杂交，构建得到SUV39H1基因条件性敲除小鼠。本发明的SUV39H1基因条件性敲除小鼠的构建方法高效、快捷、简便，得到的SUV39H1基因条件性敲除小鼠稳定性好、脱靶率低，为研究SUV39H1基因的功能和在不同生理和病理情境下的作用提供经济、可靠的动物模型。
附图说明
图1为SUV39H1基因条件性敲除小鼠的构建方法中打靶载体区域示意图；
图2为sgRNA的活性检测结果；
图3为sgRNA-4和sgRNA-10的电泳图；
图4为pCS质粒图谱。
图5为F0代小鼠基因型鉴定的引物设计原则；
图6为SUV39H1基因条件性敲除小鼠交配方案示意图；
图7为SUV39H1基因条件性敲除小鼠纯合子交配方案示意图；
图8为实施例1构建得到的SUV39H1基因条件性敲除小鼠PCR结果图；
图9为实施例1构建得到的SUV39H1基因条件性敲除小鼠Western blot分析图；
图10为实施例1构建得到的SUV39H1基因条件性敲除小鼠免疫组织化学分析图；
图11为实施例1构建得到的SUV39H1基因条件性敲除小鼠脂肪干细胞细胞的增殖、迁移速度；
图12为实施例1构建得到的SUV39H1基因条件性敲除小鼠脂肪干细胞衰老相关分泌表型改变。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1SUV39H1基因条件性敲除小鼠的构建
本实施例提供一种SUV39H1基因条件性敲除小鼠的构建方法，具体包括以下步骤：
1、sgRNA的设计
本发明所述的小鼠SUV39H1基因位于X染色体反义链上，全长13.6kb，在NCBI的基因ID为20937，基于CRISPR SgRNA的设计原则，同时考虑到小鼠SUV39H1基因的结构，发现共有5个转录本，最终选择了SUV39H1-203进行设计，其中exon2-3可被条件性敲除。5’端和3’端LoxP位点分别插入在intron1中和intron3中的非保守区；5’端和3’端的同源臂约为1.5kb。本实施例设计了14个不同的打靶位点，敲除区域示意图如图1所示，具体的sgRNA序列如表1所示。
表1sgRNA的序列
对表1的sgRNA进行活性检测，具体为构建sgRNA及Cas9表达质粒并转染细胞，筛选阳性混合克隆，挑选并培养单克隆，使用PCR实验检测编辑效率，鉴定敲除类型。检测结果如图2所示，并优选sgRNA-4和sgRNA-10进行下一步实验。
sgRNA的RNA制备：将sgRNA-4和sgRNA-10连入带T7启动子质粒载体上并进行体外转录，得到用于显微注射的RNA，并进行PCR验证，在2％琼脂糖凝胶中65℃电泳5min，结果如图3所示。
2、打靶载体构建
所述打靶载体购自上海泽叶生物科技有限公司。
3、移植注射获得阳性鼠：
将sgRNA序列合成oligos，通过退火聚合的方式连入pCS载体得到pCS质粒，所述pCS质粒的图谱如图4所示。将pCS质粒和打靶载体显微注射到小鼠受精卵中，移植得到假孕母鼠的输卵管内。待假孕母鼠所生的F0代小鼠长至一周龄，剪2mm左右鼠尾，提取鼠尾基因组进行基因型鉴定。
根据插入Floxed的两个位点设计引物，引物设计原则如图5所示。引物信息见表2，PCR反应条件和反应体系如下表3所示。
表2鉴定引物信息
表3PCR反应条件
4、筛选阳性F0代小鼠与野生型小鼠交配获得F1代小鼠，F1代小鼠经过鉴定筛选出阳性杂合子，鉴定方案与F0代小鼠相同。
5、将F1代阳性杂合子小鼠与Cre-deleter小鼠交配，如图6所示，得杂合子小鼠(SUV39H1基因条件性敲除小鼠，基因型为△/+，Cre/+)，并将其相互交配，如图7所示，得到纯合子小鼠(基因型为+/+，Cre/+)。所述SUV39H1基因条件性敲除小鼠的基因型鉴定策略如表4所示，所述纯合子小鼠的基因型鉴定策略如表5所示。
表4
表5
实施例2
本实施例对采用实施例1构建得到的SUV39H1基因条件性敲除小鼠进行验证，具体实验方法如下：
将CAG-Cre小鼠与SUV39H1-Flox小鼠杂交，获得CAG-SUV39H1条件性敲除小鼠，鉴定方法通实施例1的步骤5。对比发现CAG-SUV39H1条件性敲除小鼠的生存状况、健康程度、生殖率与对照小鼠没有区别。
选取较易获取的脂肪组织和脂肪来源的干细胞进行后续的实验验证CAG-SUV39H1条件性敲除小鼠的敲除效率，具体如下：
1、PCR验证：采用如下引物及条件对CAG-SUV39H1条件性敲除小鼠进行PCR验证，结果如图8所示。其中PCR程序为：95℃ 5min；95℃ 10s，60℃ 20s，72℃ 20s，40cycles；95℃5s；65℃ 1min；4℃∞。PCR引物具体：Forward：GCAGTGTGTGCTGTAAATCT；Reverse：ATACCCACGCCACTTAACCA。
由图7可知，CAG-SUV39H1条件性敲除小鼠的脂肪间充质干细胞中的SUV39H1表达效率下降。
2、Western blot分析：提取CAG-SUV39H1条件性敲除小鼠的DNA进行Western blot检测，结果如图9所示。，
3、免疫组织化学技术分析：采用免疫组织化学技术分析CAG-SUV39H1条件性敲除小鼠的SUV39H1蛋白表达水平，结果如图10所示。
4、检测CAG-SUV39H1条件性敲除小鼠与对照小鼠的脂肪干细胞细胞增殖、迁移速率，结果如图11所示。所述对照小鼠为CAG-SUV39H1小鼠的同窝对照小鼠。由图11可知，敲除了SUV39H1可以使脂肪间充质干细胞的增殖和迁移能力下降。
5、检测实施例1构建得到的SUV39H1基因条件性敲除小鼠与对照小鼠的脂肪干细胞细胞衰老相关分泌表型改变，结果如图12所示。由图12可知，构建的CAG-SUV39H1小鼠可以使脂肪间充质干细胞的SASP表达水平发生改变。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (5)
1.一种靶向小鼠SUV39H1基因的sgRNA组合，其特征在于，所述sgRNA组合包括5’Guide序列和3’Guide序列；所述5’Guide序列如SEQ ID NO.4所示；所述3’Guide序列如SEQ IDNO.10所示；所述sgRNA组合的识别位点分别位于SUV39H1基因的第2号外显子之前和第3号外显子之后。
2.一种SUV39H1基因条件性敲除小鼠的构建方法，其特征在于，包括以下步骤：
(1)设计筛选靶向小鼠SUV39H1基因的sgRNA组合；
(2)将sgRNA组合、Cas9蛋白和打靶载体通过显微注射到小鼠受精卵中，并将该受精卵移植至假孕小鼠，对假孕小鼠所生的仔鼠进行基因型鉴定，得F0代小鼠；
(3)将F0代小鼠与野生型小鼠交配，得F1代小鼠；
(4)经基因型鉴定筛选F1代杂合子小鼠与Cre工具鼠交配，得所述SUV39H1基因条件性敲除小鼠；
所述步骤(1)中的sgRNA的核苷酸序列如SEQ ID NO.4和SEQ ID NO.10所示。
3.根据权利要求2所述的SUV39H1基因条件性敲除小鼠的构建方法，其特征在于，所述F0代和F1代小鼠的基因型鉴定使用的5’端鉴定引物如5’loxP-F：TGTCCTATGCCAGGAATTCGCCTAA和5’loxP-R：AGCCTACACACCAGGAAGCATCAAC所示；3’端鉴定引物如3’loxP-F：AGGTCTGGGCATAAGGTTGCAGAAC和3’loxP-R：GGGGTCATTCTATCCCCAAAGGGGT所示。
4.根据权利要求2所述的SUV39H1基因条件性敲除小鼠的构建方法，其特征在于，所述F0代和F1代小鼠的基因型鉴定时，PCR扩增的程序为94℃预变性5min；98℃变性30s，67℃退火30s，68℃延伸1kb/min，每个循环退火温度降低0.7℃，共15个循环；98℃变性30s，57℃退火30s，68℃延伸1kb/min，25个循环；68℃延伸10min。
5.权利要求2-4任一项所述构建方法得到的SUV39H1基因条件性敲除小鼠在研究SUV39H1基因相关功能和作用机制中的应用。
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