WO2020000464A1 - 一种制备gl-r基因敲除小鼠的方法 - Google Patents

一种制备gl-r基因敲除小鼠的方法 Download PDF

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WO2020000464A1
WO2020000464A1 PCT/CN2018/093874 CN2018093874W WO2020000464A1 WO 2020000464 A1 WO2020000464 A1 WO 2020000464A1 CN 2018093874 W CN2018093874 W CN 2018093874W WO 2020000464 A1 WO2020000464 A1 WO 2020000464A1
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gene
crispr
sgrna
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毛吉炎
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深圳市博奥康生物科技有限公司
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  • the invention relates to the fields of genetic engineering and gene editing, and in particular, to editing a GL-R gene by using a CRISPR-Cas9 system, and obtaining a GL-R gene knockout mouse through a somatic cell nuclear transfer technology.
  • Diabetes is a group of chronic non-infectious and metabolic diseases that are characterized by increased blood glucose levels and seriously endanger human health. Whether in developed or developing countries, diabetes has become an epidemic in modern society and has become a worldwide public health problem that seriously threatens human health. According to the recommendations of the World Health Organization (WHO) and the International Diabetes Federation (IDF) expert group, diabetes can be divided into four types: type 1, type 2, other special types, and gestational diabetes, of which type 2 diabetes (T2DM) is the main body of the diabetic population . In recent years, the prevalence of type 2 diabetes has increased dramatically in various countries around the world, and the surge in patients with type 2 diabetes is the main reason for the dramatic increase in the number of diabetes patients worldwide.
  • WHO World Health Organization
  • IDF International Diabetes Federation
  • Glucagon receptor is widely distributed in various tissues and organs of the human body but is mainly located on the surface of hepatocyte membrane. It is a family of G protein coupled receptor (GPCR) superfamily. B is a typical representative of a seven-pass transmembrane protein, and its extracelluar domain (ECD) is the main domain that binds to glucagon. Many studies now believe that the excessively high glucagon concentration in the human body is an important factor that promotes the development of diabetes, and is even considered to be the central link in the pathophysiology of type 2 diabetes.
  • GPCR G protein coupled receptor
  • CRISPR-Cas9 is an adaptive immune system found in bacteria and archaea. Using artificially synthesized sgRNA sequences that are complementary to genomic DNA bases, the Cas9 enzyme can achieve site-specific cleavage of the genome, resulting in double-strand breaks in the DNA. Subsequently, the cell's DNA repair mechanism is activated, generating random types at the double-strand breaks. Insertion / deletion repair may cause gene frameshift mutation and loss of gene function, which can be used to study the function of GL-R gene.
  • an object of the present invention is to provide a method for preparing a GL-R gene knockout mouse.
  • the present invention first provides an sgRNA that specifically targets the GL-R gene, the nucleotide sequence of which is 5'-CTGGCTTACGTCCAGGCCCG-3 '.
  • the present invention also provides a CRISPR-Cas9 targeting vector containing the aforementioned sgRNA.
  • a CRISPR-Cas9 targeting vector containing the aforementioned sgRNA.
  • it is a px-459 plasmid to which sgRNA is ligated.
  • the px-459 plasmid is purchased from Addgene, and its map is shown in FIG. 1.
  • the CRISPR-Cas9 targeting vector is prepared by the following method: sgRNA and its complementary oligonucleotide sequence are synthesized by means of synthetic primers. Anneal the oligonucleotide sequence by boiling 5 min, then let stand and let it cool to room temperature. The px-459 plasmid was digested with Bbs I, and the vector backbone was recovered by digestion. T4 DNA ligase was used to ligate with the annealed oligonucleotide product.
  • the present invention also provides a method for preparing a GL-R gene knockout cell, which transfects a CRISPR-Cas9 targeting vector containing an sgRNA targeting the CDS region of the GL-R gene, thereby knocking out the cell's GL- R gene.
  • the present invention also provides a method for preparing a GL-R gene knockout mouse, that is, the GL-R gene is knocked out by using a CRISPR-Cas9 targeting vector targeting the GL-R gene CDS region.
  • the method includes the following steps:
  • step 2 co-transfect the linearized fragment obtained in step 1 into mouse embryonic fibroblasts, and select single-cell clones with resistance by puromycin;
  • the CRISPR-Cas9 targeting vector px-459 was used to transduce mouse embryonic fibroblasts by taking 3 ⁇ g of the px-459 plasmid and transfecting it with a lonza nuclear electrophoresis apparatus and a fibroblast electrotransformation kit.
  • the sgRNA designed for the CDS region of the mouse GL-R gene for the first time is cut by the GL-R gene of the CRISPR-Cas9 system, and a GL-R knockout mouse individual is obtained.
  • This preparation of the GL-R gene The method of knocking out mice has not been reported before at home and abroad. It provides a practical method for studying mouse GL-R gene.
  • Figure 1 shows the px-459 vector map.
  • px-459 vector was purchased from Addgene company, T4 DNA ligase, Bbs I and T7E1 were purchased from NEB company, and high-fidelity PCR enzyme was purchased from Dalian Bio-Bio. Primer synthesis and sequencing were completed by Shanghai Shengong, endotoxin-free plasmid extraction kit The gel recovery kit and genomic extraction kit were purchased from QIAGEN.
  • Embodiment one CRSIPR-Cas9 Construction of a targeting vector
  • sgRNA was designed in the CDS region of mouse GL-R gene, and its sequence was 5 '-CTGGCTTACGTCCAGGCCCG -3'. According to the principle of base complementary pairing, its reverse complementary sequence was 5'- CGGGCCTGGACGTAAGCCAG. -3 '.
  • the px-459 vector backbone needs to be digested with BbsI, so the sticky end of the BbsI digestion site needs to be added to the sgRNA sequence to facilitate its integration into the pX330 vector backbone. Add the sgRNA sequence of BbsI sticky ends and its complementary sequence.
  • the designed sgRNA added to the cohesive end of the BbsI digestion site and its complementary sequence are synthesized by means of synthetic primers.
  • the synthetic oligonucleotide is annealed to form a double-stranded DNA with sticky ends. The annealing procedure is: boil for 5 min, then let it stand and allow it to cool naturally to room temperature.
  • the px-459 vector backbone was digested with Bbs I and reacted at 37 ° C for 2 h. Then perform agarose gel electrophoresis and cut the gel to recover the target band.
  • the vector backbone is linked to the sgRNA sequence to construct a targeting vector.
  • the recovered vector backbone was ligated with the sgRNA sequence annealing product at 4 ° C overnight.
  • the ligated product was transformed into Top10 competent cells, cultured in an incubator at 37 ° C, and when a monoclonal was grown, a single line was picked and sequenced to identify a positive monoclonal.
  • the target vector was extracted without endotoxin extraction in the expanded culture.
  • Example 3 Transduction of mouse embryonic fibroblasts and screening of single cell clones
  • mouse embryonic fibroblasts whose fusion degree has reached 80-90% are digested and centrifuged to obtain about 200,000-2,000,000 mouse embryonic fibroblasts.
  • the cells in the cell culture dish should adhere and be in good condition. Add 1-3 ⁇ g / ml puromycin, and then add puromycin every 2 days. The dosage should be based on the cell status and Convergence is flexible. After screening for 7-10 days, it can be seen that the monoclonal growth.
  • Embodiment 5 GL-R Preparation of knockout mice
  • Example 4 Use the positive mouse embryonic fibroblasts obtained in Example 4 as the nuclear transfer donor cells. Culture embryo fibroblasts to 100% confluence for 1-2 days, remove the culture medium in the dish, wash once with PBS, and then digest with 0.1% trypsin for about 2 minutes. Immediately after the cells become round, use 10% FBS. The digestion of the cell culture medium was terminated, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells pelleted by centrifugation were resuspended, and placed in an ice bath for later use.
  • In vitro mature oocytes are used as nuclear transfer recipient eggs. After artificially inducing ovulation in female mice, cumulus-oocyte complexes were collected from their ovaries, matured in vitro and hyaluronidase was used to remove cumulus cells, and then the first polar body and morphology were selected and discharged under a stereomicroscope. Normal, cytoplasmic mature oocytes are ready for use.
  • mice Use an embryo transfer tube to aspirate the embryo and place it into the joint around the fallopian tube bell mouth, and then reset the fat pad, ovary, fallopian tube, and uterus to suture the wound.
  • the mice were bred in a single cage until delivery after awakening.
  • the sgRNA designed for the CDS region of the mouse GL-R gene for the first time is cut by the GL-R gene of the CRISPR-Cas9 system, and a GL-R knockout mouse individual is obtained.
  • This preparation of the GL-R gene The method of knocking out mice has not been reported before at home and abroad. It provides a practical method for studying mouse GL-R gene.

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Abstract

利用CRISPR-Cas9系统对GL-R基因进行编辑，并通过体细胞核移植技术获得GL-R基因敲除小鼠。首次针对小鼠GL-R基因的CDS区设计sgRNA，并利用CRISPR-Cas9系统对其进行切割，实现了GL-R基因的敲除，并且获得了相应的敲除小鼠个体，为研究小鼠GL-R基因提供了一种切实可行的方法。

Description

一种制备GL-R基因敲除小鼠的方法 技术领域
本发明涉及基因工程和基因编辑领域，具体地说，涉及利用CRISPR-Cas9系统对GL-R基因进行编辑，并通过体细胞核移植技术获得GL-R基因敲除小鼠。
 背景技术
糖尿病是一组以血葡萄糖水平增高为特征的、严重危害人类健康的慢性非传染性、代谢性疾病。无论是在发达国家还是发展中国家，糖尿病成为现代社会的流行病，已成为严重威胁人类健康的世界性公共卫生问题。按照世界卫生组织（WHO）及国际糖尿病联盟（IDF）专家组的建议，糖尿病可分为1型、2型、其他特殊类型及妊娠糖尿病4种，其中2型糖尿病（T2DM）是糖尿病人群的主体。近年来，世界各国2型糖尿病的患病率均有急剧增加的趋势，2型糖尿病患者激增是造成全世界糖尿病患者总数剧增的主要原因。据近年大量流行病学调查结果显示，糖尿病在成人中的患病率已接近10%，其中2型糖尿病占全体糖尿病患者的90%左右，且近三、五十年内2型糖尿病急剧增加的趋势仍将继续，防治形势十分严峻。
 技术问题
胰高血糖素的受体(Glucagon receptor，GL-R)在人体各种组织器官中广泛分布但主要位于肝细胞膜表面，是G蛋白偶联受体(G protein coupled receptor，GPCR)超家族中家族B的典型代表，属于七次跨膜蛋白，其胞外区(Extracelluar domain，ECD)是与胰高血糖素结合的主要结构域。现在有很多研究认为人体内胰高血糖素浓度过高是促使糖尿病发生发展的重要因素，甚至被认为是2型糖尿病病理生理过程中的中心环节。
CRISPR-Cas9是一种存在于细菌和古生菌中的适应性免疫系统。利用人工合成的、与基因组DNA碱基互补配对的sgRNA序列，Cas9酶可以实现基因组的定点切割，从而产生DNA的双链断裂，随后细胞的DNA修复机制被激活，在双链断裂处产生随机类型的插入/缺失修复，可能会造成基因的移码突变和基因功能缺失，即可用于GL-R基因功能的研究。
 技术解决方案
为了解决现有技术中存在的问题，本发明的目的是提供一种制备GL-R基因敲除小鼠的方法。
为了实现本发明目的，本发明首先提供特异性靶向GL-R基因的sgRNA，其核苷酸序列为5’ - CTGGCTTACGTCCAGGCCCG -3’。
    其次，本发明还提供了含有前述sgRNA的CRISPR-Cas9打靶载体。作为优选，其为连接有sgRNA的px-459质粒，所述px-459质粒购自Addgene，其图谱如图1所示。
    所述CRISPR-Cas9打靶载体，是通过以下方法制备得到的：以合成引物的方式，合成sgRNA及其互补的寡核苷酸序列。将寡核酸序列进行退火操作，方法为煮沸5 min，然后静置并使其自然冷却至室温。用Bbs I酶切px-459质粒，切胶回收载体骨架，利用T4 DNA连接酶与退火后的寡核苷酸产物进行连接。
进一步地，本发明还提供了一种制备GL-R基因敲除细胞的方法，将含有靶向GL-R基因CDS区的sgRNA的CRISPR-Cas9打靶载体转染细胞，从而敲除细胞的GL-R基因。
与此同时，本发明还提供了一种制备GL-R基因敲除小鼠的方法，即同时利用靶向GL-R基因CDS区的CRISPR-Cas9打靶载体，对GL-R基因实现敲除。
具体而言，所述方法包括如下步骤：
① 将含有靶向GL-R基因CDS区的sgRNA的CRISPR-Cas9打靶载体进行酶切，得到线性化片段；
② 将步骤①得到的线性化片段共转染小鼠的胚胎成纤维细胞，通过嘌呤霉素筛选具有抗性的单细胞克隆；
③ 选取状态良好的阳性单细胞克隆作为核移植的供体细胞，卵母细胞作为核移植的受体细胞，利用体细胞核移植技术构建克隆胚胎，将优质的克隆胚胎移植到代孕小鼠的输卵管内，经过全期发育获得GL-R基因敲除小鼠。
其中，CRISPR-Cas9打靶载体px-459转导小鼠的胚胎成纤维细胞的方法为：取px-459质粒3 μg，利用lonza核电转仪及成纤维细胞电转试剂盒进行转染。
 有益效果
本发明首次针对小鼠GL-R基因的CDS区设计的sgRNA，并借助CRISPR-Cas9系统GL-R基因实现了切割，获得了GL-R敲除的小鼠个体，这种制备GL-R基因敲除小鼠的方法在国内外之前是没有报道的。为研究小鼠GL-R基因提供了一种切实可行的方法。
 附图说明
图1为px-459载体图谱。
 本发明的实施方式
下面结合具体实施例和附图进一步阐述本发明。
px-459载体购自Addgene公司，T4 DNA连接酶、Bbs I及T7E1购于NEB公司，高保真PCR酶购自大连宝生物，引物合成及测序由上海生工完成，去内毒素质粒提取试剂盒、胶回收试剂盒及基因组提取试剂盒购于QIAGEN公司。
实施例一： CRSIPR-Cas9 打靶载体的构建
根据CRISPR-Cas9的作用原理，在小鼠GL-R基因的CDS区设计sgRNA，其序列为5’ - CTGGCTTACGTCCAGGCCCG -3’， 根据碱基互补配对的原则，其反向互补序列为5’- CGGGCCTGGACGTAAGCCAG -3’。
px-459载体骨架需要使用BbsⅠ进行酶切，所以需要在sgRNA序列上补出BbsⅠ酶切位点的粘性末端，以利于其连入pX330载体骨架。加入BbsⅠ 粘性末端的sgRNA序列及其互补序列。
a. 将设计好的加入BbsⅠ酶切位点粘性末端的sgRNA及其互补序列以合成引物的方式进行合成。将合成的寡核苷酸进行退火操作，使其形成带有粘性末端的DNA双链。退火程序为：煮沸5 min，然后静置并使其自然冷却至室温。
b. px-459载体骨架使用BbsⅠ酶切，37℃反应2 h。然后进行琼脂糖凝胶电泳，并切胶回收目的条带。
c. 载体骨架与sgRNA序列连接，构建打靶载体。将回收的载体骨架与sgRNA序列退火产物于4℃连接过夜。将连接产物转化Top10感受态细胞，37℃培养箱培养，待其长出单克隆后 ，挑取单克隆划线，并进行测序鉴定阳性单克隆。对于测序结果正确的阳性克隆，扩大培养并无内毒素提取其中的打靶载体。
实施例二：小鼠胚胎成纤维细胞的建系
a. 取妊娠10-15天的小鼠，从其子宫内无菌取出胚胎，置于超净工作台中，用眼科剪去除胚胎的头部、四肢、内脏及软骨组织，用PBS冲洗干净；
b. 在细胞培养皿内用眼科剪将剩余组织剪碎成约1平方毫米小块；
c. 加入适量的FBS，保持组织不至于过分干燥。将剪碎的组织块转移到1个T75细胞培养瓶中，将组织块均匀铺开；
d. 加入5 ml细胞培养基，将铺有组织块的一面向上，不被培养基浸没，于37℃，5％CO2培养箱中培养3～5 h后，将培养瓶翻转，使组织块被培养基浸没；
e. 培养5天左右，观察到组织块周围有大量细胞爬出，待细胞生长至约90％汇合度时，对细胞进行消化并冻存备用。
实施例三：小鼠胚胎成纤维细胞的转导和单细胞克隆的筛选
a. 在一个六孔板孔中，将融合度已达到80-90％的小鼠胚胎成纤维细胞，进行消化、离心，获得数量约200000-2000000的小鼠胚胎成纤维细胞。
b. 将打靶载体加入Lonza转染试剂中，混匀。使用加入质粒的转染试剂重悬细胞，并将细胞悬液加入到电击杯中，开始电击细胞。
c. 电击完成后，立即将细胞吸出，加2 ml含10％ FBS的DMEM到六孔板中。
d. 37℃，5％ CO2培养箱培养48h后，细胞达到80％-90％汇合，将细胞消化下来，稀释至20-30个100 mm细胞培养皿中。
e. 24-48h后，待细胞培养皿中的细胞贴壁、且状态良好，加入1-3 μg/ml的嘌呤霉素，每2天补加一次嘌呤霉素，加药量根据细胞状态及汇合度灵活掌控。筛选7-10天，可见单克隆长出。
f. 单克隆的挑取及扩大培养。在显微镜下，使用记号笔将状态良好的单克隆用圆圈圈出。弃掉培养皿中的培养基，PBS清洗一次，将克隆环蘸取明胶，用克隆环将细胞单克隆圈住，加入10-30 μl 0.1％的胰蛋白酶，37℃消化1 min。在显微镜下观察，待细胞变圆、游离时，立即加入含10％ FBS的DMEM终止消化。将细胞吸出加入24孔板中。48-72h后，24孔板中细胞融合度达到80-90％时，将细胞传至12孔板中。待12孔板中细胞融合度达到80％-90％时，对细胞进行冻存。
实施例四：阳性单细胞克隆的鉴定
取30个单克隆的基因组DNA，以其为模板进行PCR扩增，,以野生型细胞的基因组作为阴性对照，并使用T7E1酶对PCR产物进行酶切鉴定，筛选出酶切结果为阳性的单克隆。
实施例五： GL-R 基因敲除小鼠的制备
a. 以实施例四获得的阳性小鼠胚胎成纤维细胞为核移植供体细胞。培养胚胎成纤维细胞至100％融合1-2天，去除培养皿内培养基，加入PBS洗涤1次，然后用0.1％胰蛋白酶消化约2 min，待细胞变圆后立即后用含10% FBS的细胞培养液终止消化，1000 rpm离心5min，弃上清，重悬离心沉淀的细胞，冰浴放置备用。
b. 以体外成熟的卵母细胞为核移植受体卵质。人工诱导雌性小鼠排卵后，从其卵巢中采集取卵丘-卵母细胞复合体，经过体外成熟并用透明质酸酶脱去卵丘细胞，而后在体式显微镜下挑选排出第一极体、形态正常、胞质均匀的成熟卵母细胞备用。
c. 在显微操作仪下，将核移植供体细胞移入去核的成熟卵母细胞中。经过电融合及化学激活，诱导细胞与卵子融合并同时激活卵母细胞。构建成重组胚胎，融合胚放入低氧培养环境下培养。体外发育至1-4细胞期后观察卵裂情况及发育状态，并用于胚胎移植。
d. 挑选形态正常、发育优良的克隆胚胎移植入胚胎同期的雌性小鼠体内。将假孕的雌性小鼠麻醉后，沿背腰中央纵向剪开皮肤，用镊子沿切口剥开皮肤、肌肉，找到脂肪垫。在脂肪垫的位置剪开肌肉层，用小镊子夹住脂肪组织，把一侧的卵巢、输卵管与子宫一起拉出，固定好，置于显微镜下。使用胚胎移植管吸取胚胎并将其放入输卵管喇叭口周围接合部，然后将脂肪垫、卵巢、输卵管及子宫等复位，缝合伤口。小鼠苏醒后单笼饲养至分娩。
 工业实用性
本发明首次针对小鼠GL-R基因的CDS区设计的sgRNA，并借助CRISPR-Cas9系统GL-R基因实现了切割，获得了GL-R敲除的小鼠个体，这种制备GL-R基因敲除小鼠的方法在国内外之前是没有报道的。为研究小鼠GL-R基因提供了一种切实可行的方法。

Claims (6)

  1. 特异性靶向GL-R基因的sgRNA，其特征在于，其核苷酸序列为5’ – CTGGCTTACGTCCAGGCCCG -3’。
  2. 含有权利要求1所述的sgRNA的CRISPR-Cas9打靶载体。
  3. 根据权利要求2所述的CRISPR-Cas9打靶载体，其特征在于，其为连接有sgRNA的px-459质粒。
  4. 一种制备GL-R基因敲除细胞的方法，其特征在于，将含有权利要求2所述sgRNA的CRISPR-Cas9打靶载体转染细胞，从而敲除细胞的GL-R基因。
  5. 一种制备GL-R基因敲除小鼠的方法，其特征在于，利用靶向GL-R基因CDS区的CRISPR-Cas9打靶载体，对GL-R基因实现敲除。
  6. 根据权利要求7所述的方法，其特征在于，包括如下步骤：
    ① 将含有权利要求1所述sgRNA的CRISPR-Cas9打靶载体进行酶切，得到线性化片段；
    ② 将步骤①得到的线性化片段共转染小鼠的胚胎成纤维细胞，通过嘌呤霉素筛选具有抗性的单细胞克隆；
    ③ 选取状态良好的阳性单细胞克隆作为核移植的供体细胞，卵母细胞作为核移植的受体细胞，利用体细胞核移植技术构建克隆胚胎，将优质的克隆胚胎移植到代孕母小鼠的输卵管内，经过全期发育获得GL-R基因敲除小鼠。
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