WO2021243881A1 - Sgrna composition for knocking out porcine mbp gene and use thereof - Google Patents

Sgrna composition for knocking out porcine mbp gene and use thereof Download PDF

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WO2021243881A1
WO2021243881A1 PCT/CN2020/114926 CN2020114926W WO2021243881A1 WO 2021243881 A1 WO2021243881 A1 WO 2021243881A1 CN 2020114926 W CN2020114926 W CN 2020114926W WO 2021243881 A1 WO2021243881 A1 WO 2021243881A1
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expression vector
sgrna
mbp gene
composition
cell
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唐成程
邹庆剑
周小青
陈敏
赖良学
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五邑大学
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Definitions

  • the invention belongs to the field of gene editing, and relates to an sgRNA composition for pig MBP gene knockout and its use.
  • mice There is now a very commonly used model mouse for myelin deficiency disease called shiverer mouse.
  • This model mouse has a large fragment of MBP (Myelin basic protein) gene deletion, that is, from intron 1 to exon. The gene of exon 6 is deleted, leaving only the intron sequence including exon 1 and 12.4-14.4 kb. Due to the deletion of a large fragment of the MBP gene, the myelin sheath of the central nervous system has no myelin cells.
  • MBP Myelin basic protein
  • This kind of mice is heterozygous and normal, and homozygous mice generally begin to tremble about 12 days after birth, and their symptoms intensify as they age, and usually die in about 50-100 days. At present, it is widely used in neurobiology, research on related diseases such as myelin loss.
  • mice Because the size and nervous system of mice are quite different from those of humans, they cannot well mimic the characteristics of human related diseases. Pigs are more similar to humans in body size, and there are articles showing that pigs have an advantage over mice in simulating human nervous system diseases. Therefore, we plan to use efficient CRISPR/Cas9 gene editing technology and somatic cell nuclear transfer technology to prepare a pig model of myelin loss, and then use this model as a basis for cell therapy, hoping to lay the foundation for cell therapy and gene therapy of related diseases.
  • the purpose of the present invention is to provide an efficient and simple method to obtain large MBP gene fragment knockout porcine fibroblasts, and the present invention also provides corresponding preparation methods and detection means.
  • the technical solution adopted by the present invention is: a sgRNA composition for pig MBP gene knockout, the composition comprising sgRNA1 and sgRNA2; the nucleotide sequence of the sgRNA1 and sgRNA2 ranges from 5'to 3' The directions are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • the present invention also claims the application of the sgRNA composition in the construction of a sgRNA expression vector composition for knocking out the pig MBP gene.
  • the present invention also claims a sgRNA expression vector composition for knocking out pig MBP genes, said expression vector comprising expression vector 1 and expression vector 2; 1. said expression vector comprising expression vector 1 and expression vector 2; said expression Vector 1 contains the sequence shown in SEQ ID NO.1, and the expression vector 2 contains the sequence shown in SEQ ID NO.2.
  • the vector backbone of the expression vector composition is pX459.
  • the present invention also claims a method for constructing the expression vector composition, which includes the following steps:
  • step (3) Connect the double strands of step (3) to the digested products of step (2) respectively, and transform and identify them to obtain the expression vector composition.
  • the sense strand and antisense strand of the sgRNA contain sequences corresponding to the restriction site of the vector, which are complementary to the ends of the vector after restriction, so as to be connected to the vector.
  • the backbone is digested by BbsI, and the size of the digested product is about 9162bp; the sense strand and antisense strand corresponding to sgRNA1 are shown in SEQ ID NO.3 and SEQ ID NO.4, and sgRNA2 corresponds to the sense The chain and the antisense strand are shown in SEQ ID NO. 5 and SEQ ID NO. 6 (containing the BbsI digestion sequence respectively), forming a double-stranded DNA fragment with BbsI sticky ends connected to the digested product.
  • the present invention also claims the use of the sgRNA composition and the expression vector composition in the preparation of MBP gene knockout cell lines or animal models.
  • the cells are porcine fibroblasts.
  • the present invention also claims a method for preparing MBP gene knockout cells, the method comprising transferring the expression vector composition into the cells.
  • the carrier used does not contain the Cas9 protein, it is necessary to carry out the transfection of the Cas9 protein carrier at the same time, or use a pre-built cell stably expressing the Cas9 protein.
  • the preferred vector pX459 of the present invention contains the coding sequence of Cas9, which can express Cas9 protein and sgRNA after being transformed into cells, so as to achieve the purpose of gene knockout.
  • the method for transferring the expression vector composition into cells is electroporation.
  • the method further includes screening for resistant cells.
  • the vector contains the corresponding resistance gene (not contained in certain wild-type cells)
  • the cells containing the relevant gene that is, successfully transferred into the vector
  • the vector pX459 contains a puromycin resistance marker, and an appropriate concentration of puromycin can be used for cell selection.
  • the method further includes the culturing of monoclonal cells of the target cells.
  • the knockout of the MBP gene in each single clone can be identified.
  • the present invention also claims to protect the MBP gene knockout cells prepared by the method.
  • the cells are porcine fibroblasts.
  • the porcine fibroblasts can be used to prepare transgenic cloned pigs.
  • the present invention also claims the use of an MBP gene knockout cell or animal model in drug screening for treating diseases caused by MBP gene defects, and the MBP gene knockout cell or animal model is combined by the expression vector ⁇ Preparation obtained.
  • the cell or animal model is a homozygous cell or animal model containing a lack of a large fragment of the MBP gene.
  • the method of the present invention adopts two sgRNA sequences respectively targeting exon 1 and exon 6 of pig MBP gene, and large fragment gene knockout cells can be obtained through screening (preferably through single cell screening)
  • This method is simple to operate, and is more efficient than traditional gene modification methods such as Cre-loxP (shorter time) and microinjection (no chimerism).
  • the pig MBP gene knockout cell line obtained by this method can be directly used for somatic cell cloning to obtain genetically modified animal pigs. Because pigs have more advantages than mice in terms of body size and simulation of human nervous system, they will be used in the future for related diseases. Play a greater role in the study of pathogenic mechanisms and disease treatment options.
  • Figure 1 shows the results of Sanger sequencing of porcine fibroblasts with MBP gene knocked out constructed by the method of the present invention.
  • Example 1 sgRNA composition and its expression vector for pig MBP gene knockout
  • the Cas9 target site contains 20 bases, and the 3 bases immediately adjacent to the 3'end of the target constitute the PAM region.
  • the PAM region requires a sequence of NGG (N is any base). Designed according to this principle, one sgRNA was designed for the first exon and the sixth exon of the pig MBP gene, and the sequence from the 5'-3' direction is as follows:
  • the sgRNA sequence designed for the first exon GAGGCACGGATCAAAGTACC (SEQ ID NO.1); the corresponding sense strand sequence and antisense strand sequence are:
  • the sgRNA sequence designed for exon 6 GAGGACGGGACAGCCGCTCC (SEQ ID NO. 2); the corresponding sense strand sequence and antisense strand sequence are:
  • the bold part includes the BbsI restriction site.
  • the backbone plasmid is PX459 V2.0 (addgene,62988).
  • the digestion system is as shown in Table 1. Digestion at 37°C for about 5 hours. Before recovery, take 5 ⁇ L of the product and run the gel to confirm that the digestion is complete.
  • the digested product (about 9162 bp) was recovered using the gel recovery kit (TIANGEN).
  • the annealing procedure is: 37°C for 30 minutes, 95°C for 5 minutes, and then the temperature is reduced to 25°C at a rate of 5°C/min.
  • step (1) The product recovered by digestion of step (1) 50ng
  • the oligos of step (2) (diluted 200 times) 1 ⁇ L T4 Ligase(NEB) 0.5 ⁇ L 10 ⁇ T4 Ligation Buffer(NEB) 1 ⁇ L ddH 2 O Make up to 10 ⁇ L
  • porcine fibroblasts grow to about 80%, they are passaged at 1:10; 24 hours later, fresh media containing different concentrations of puromycin are added (at least 5 concentration gradients are set within 0-2 ⁇ g/mL). Observe the cell survival ratio under the microscope every day (the best action time of puromycin is generally 1-4 days), and select the lowest concentration of puromycin that kills all cells for 2-3 days as the concentration of puromycin used for cell selection .
  • the screening concentration of puromycin in this experiment is 800ng/ ⁇ L.
  • cell clones can be seen under the microscope.
  • the cell clones are picked by trypsinization of the cloning ring (Corning, 3166-8), and each cell clone is picked to a 24-well plate When the 24-well cell clone grows to about 90%, pass 1:2, and take out some cells for PCR and sequencing for identification.
  • the upstream primer used for identification is generally designed at 100-200bp upstream of the sgRNA site, and the downstream primer is generally designed at 100-200bp downstream of the sgRNA site.
  • the identification primers in this experiment are:
  • RP1 GGGATGTCACTGTCTCCGAGGTA.
  • the PCR amplification conditions are:

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Abstract

Provided are an sgRNA composition for knocking out a porcine myelin basic protein (MBP) gene and use thereof. The sgRNA composition comprises an sgRNA sequence separately targeted to a porcine MBP gene exon 1 and exon 6. A cell line in which a large fragment of the MBP gene is knocked out can be obtained by using a vector capable of expressing the sgRNA and a Cas9 protein.

Description

用于猪MBP基因敲除的sgRNA组合物及用途SgRNA composition for pig MBP gene knockout and use 技术领域Technical field
本发明属于基因编辑领域，涉及用于猪MBP基因敲除的sgRNA组合物及用途。The invention belongs to the field of gene editing, and relates to an sgRNA composition for pig MBP gene knockout and its use.
背景技术Background technique
现在有一种非常常用的髓鞘缺失类疾病模型鼠，叫shiverer mouse，这种模型鼠的髓鞘碱性蛋白基因即MBP(Myelin basic protein)基因大片段缺失，即从内含子1到外显子6的基因缺失，只剩包括外显子1及12.4-14.4kb的内含子序列。由于MBP基因的大片段缺失导致中枢神经系统髓鞘没有髓鞘细胞。这种小鼠，杂合正常，纯合小鼠一般出生12天左右即开始出现震颤，并且随着年龄的增长，症状加剧，一般在50-100天左右死亡。目前被广泛应用神经生物学，髓鞘缺失类相关疾病的研究。There is now a very commonly used model mouse for myelin deficiency disease called shiverer mouse. This model mouse has a large fragment of MBP (Myelin basic protein) gene deletion, that is, from intron 1 to exon. The gene of exon 6 is deleted, leaving only the intron sequence including exon 1 and 12.4-14.4 kb. Due to the deletion of a large fragment of the MBP gene, the myelin sheath of the central nervous system has no myelin cells. This kind of mice is heterozygous and normal, and homozygous mice generally begin to tremble about 12 days after birth, and their symptoms intensify as they age, and usually die in about 50-100 days. At present, it is widely used in neurobiology, research on related diseases such as myelin loss.
由于小鼠的体型及神经系统与人类相差较大，并不能很好的模拟人类的相关疾病特征。猪由于在体型上与人类更为相似，且有文章表明猪在模拟人类神经系统疾病上相较于小鼠更有优势。因此，我们拟利用高效的CRISPR/Cas9基因编辑技术和体细胞核移植技术制备髓鞘缺失猪模型，然后再以此模型为基础进行细胞治疗，希望能为相关疾病的细胞治疗和基因治疗奠定基础。Because the size and nervous system of mice are quite different from those of humans, they cannot well mimic the characteristics of human related diseases. Pigs are more similar to humans in body size, and there are articles showing that pigs have an advantage over mice in simulating human nervous system diseases. Therefore, we plan to use efficient CRISPR/Cas9 gene editing technology and somatic cell nuclear transfer technology to prepare a pig model of myelin loss, and then use this model as a basis for cell therapy, hoping to lay the foundation for cell therapy and gene therapy of related diseases.
发明内容Summary of the invention
针对上述问题，本发明的目的是提供一种高效且简单的方法获得MBP基因大片段敲除猪成纤维细胞，同时本发明还提供相应的制备方法和检测手段。In view of the above-mentioned problems, the purpose of the present invention is to provide an efficient and simple method to obtain large MBP gene fragment knockout porcine fibroblasts, and the present invention also provides corresponding preparation methods and detection means.
为实现上述目的，本发明采取的技术方案为：用于猪MBP基因敲除的sgRNA组合物，所述组合物包括sgRNA1和sgRNA2；所述sgRNA1和sgRNA2 的核苷酸序列从5’到3’方向如SEQ ID NO.1和SEQ ID NO.2所示。In order to achieve the above purpose, the technical solution adopted by the present invention is: a sgRNA composition for pig MBP gene knockout, the composition comprising sgRNA1 and sgRNA2; the nucleotide sequence of the sgRNA1 and sgRNA2 ranges from 5'to 3' The directions are shown in SEQ ID NO.1 and SEQ ID NO.2.
本发明还要求保护所述的sgRNA组合物在构建敲除猪MBP基因的sgRNA表达载体组合物中的应用。The present invention also claims the application of the sgRNA composition in the construction of a sgRNA expression vector composition for knocking out the pig MBP gene.
本发明还要求保护用于敲除猪MBP基因的sgRNA表达载体组合物，所述表达载体包括表达载体1和表达载体2；1、所述表达载体包括表达载体1和表达载体2；所述表达载体1含有如SEQ ID NO.1所示的序列，所述表达载体2含有如SEQ ID NO.2所示的序列。The present invention also claims a sgRNA expression vector composition for knocking out pig MBP genes, said expression vector comprising expression vector 1 and expression vector 2; 1. said expression vector comprising expression vector 1 and expression vector 2; said expression Vector 1 contains the sequence shown in SEQ ID NO.1, and the expression vector 2 contains the sequence shown in SEQ ID NO.2.
作为本发明的优选实施方式，所述表达载体组合物的载体骨架为pX459。As a preferred embodiment of the present invention, the vector backbone of the expression vector composition is pX459.
本发明还要求保护所述表达载体组合物的构建方法，包括如下步骤：The present invention also claims a method for constructing the expression vector composition, which includes the following steps:
(1)合成所述sgRNA对应的正义链和反义链；(1) Synthesize the sense strand and antisense strand corresponding to the sgRNA;
(2)酶切载体骨架，并回收酶切产物；(2) Enzyme digestion of the vector backbone, and recovery of the digestion product;
(3)分别将步骤(1)所述sgRNA对应的正义链和反义链进行退火得到双链；(3) Annealing the sense strand and antisense strand corresponding to the sgRNA in step (1) respectively to obtain a double strand;
(4)将步骤(3)的双链分别连接到步骤(2)的酶切产物上，转化鉴定，即得所述表达载体组合物。(4) Connect the double strands of step (3) to the digested products of step (2) respectively, and transform and identify them to obtain the expression vector composition.
所述sgRNA的正义链和反义链包含对应所述载体酶切位点的序列，与酶切后的载体末端互补，从而连入载体中。The sense strand and antisense strand of the sgRNA contain sequences corresponding to the restriction site of the vector, which are complementary to the ends of the vector after restriction, so as to be connected to the vector.
当载体骨架为pX459，骨架通过BbsI进行酶切，酶切产物的大小约为9162bp；sgRNA1对应的正义链、反义链如SEQ ID NO.3和SEQ ID NO.4所示，sgRNA2对应的正义链、反义链如SEQ ID NO.5和SEQ ID NO.6所示(分别含有BbsI酶切序列)，形成与酶切产物连接的具有BbsI粘性末端的双链DNA片段。When the vector backbone is pX459, the backbone is digested by BbsI, and the size of the digested product is about 9162bp; the sense strand and antisense strand corresponding to sgRNA1 are shown in SEQ ID NO.3 and SEQ ID NO.4, and sgRNA2 corresponds to the sense The chain and the antisense strand are shown in SEQ ID NO. 5 and SEQ ID NO. 6 (containing the BbsI digestion sequence respectively), forming a double-stranded DNA fragment with BbsI sticky ends connected to the digested product.
若使用其他载体，根据不同载体的图谱确定酶切位点、sgRNA正义链、反义链上用于连接的酶切位点序列，以及回收产物的大小。If other vectors are used, determine the restriction site, sgRNA sense strand, and antisense strand of the restriction site for ligation according to the maps of different vectors, and the size of the recovered product.
本发明还要求保护所述sgRNA组合物、所述表达载体组合物在制备MBP基因敲除的细胞系或动物模型中的用途。The present invention also claims the use of the sgRNA composition and the expression vector composition in the preparation of MBP gene knockout cell lines or animal models.
作为本发明的优选实施方式，所述细胞为猪成纤维细胞。As a preferred embodiment of the present invention, the cells are porcine fibroblasts.
本发明还要求保护制备MBP基因敲除细胞的方法，所述方法包括将所述的表达载体组合物转入细胞中。The present invention also claims a method for preparing MBP gene knockout cells, the method comprising transferring the expression vector composition into the cells.
若使用的载体上未包含Cas9蛋白，则需要同时进行Cas9蛋白载体的转染，或使用预先构建好的稳定表达Cas9蛋白的细胞。本发明优选的载体pX459载体上含有Cas9的编码序列，转入细胞后可表达Cas9蛋白以及sgRNA，从而达到基因敲除的目的。If the carrier used does not contain the Cas9 protein, it is necessary to carry out the transfection of the Cas9 protein carrier at the same time, or use a pre-built cell stably expressing the Cas9 protein. The preferred vector pX459 of the present invention contains the coding sequence of Cas9, which can express Cas9 protein and sgRNA after being transformed into cells, so as to achieve the purpose of gene knockout.
作为本发明的优选实施方式，所述将表达载体组合物转入细胞的方法为电转。As a preferred embodiment of the present invention, the method for transferring the expression vector composition into cells is electroporation.
作为本发明的优选实施方式，所述方法还包括进行抗性细胞的筛选。As a preferred embodiment of the present invention, the method further includes screening for resistant cells.
由于载体上含有相应的抗性基因(一定野生型的细胞中不含有)，通过在培养基中加入抗性基因相应的药物，可对含有相关基因(即为成功转入载体)的细胞进行筛选，从而得到更高纯度的目的细胞。优选载体pX459中含有嘌呤霉素抗性标记，可用适当浓度的嘌呤霉素进行细胞筛选。Since the vector contains the corresponding resistance gene (not contained in certain wild-type cells), by adding the drug corresponding to the resistance gene in the medium, the cells containing the relevant gene (that is, successfully transferred into the vector) can be screened , So as to obtain higher purity target cells. Preferably, the vector pX459 contains a puromycin resistance marker, and an appropriate concentration of puromycin can be used for cell selection.
作为本发明的优选实施方式，所述方法还包括目标细胞的单克隆细胞的培养。As a preferred embodiment of the present invention, the method further includes the culturing of monoclonal cells of the target cells.
通过调整细胞培养密度，可将得到筛选后的具有单一基因型的单克隆细胞。By adjusting the cell culture density, monoclonal cells with a single genotype can be obtained after screening.
进一步地，通过提取单克隆细胞的DNA进行测序，可鉴别各单克隆中MBP基因的敲除情况。Furthermore, by extracting the DNA of the single clones for sequencing, the knockout of the MBP gene in each single clone can be identified.
本发明还要求保护所述方法制备的MBP基因敲除细胞。The present invention also claims to protect the MBP gene knockout cells prepared by the method.
作为本发明的优选实施方式，所述细胞为猪成纤维细胞。As a preferred embodiment of the present invention, the cells are porcine fibroblasts.
所述猪成纤维细胞可用于制备转基因克隆猪。The porcine fibroblasts can be used to prepare transgenic cloned pigs.
本发明还要求保护一种MBP基因敲除的细胞或动物模型在用于治疗MBP基因缺陷引起的疾病的药物筛选中的用途，所述MBP基因敲除的细胞或动物模 型通过所述表达载体组合物制备得到。The present invention also claims the use of an MBP gene knockout cell or animal model in drug screening for treating diseases caused by MBP gene defects, and the MBP gene knockout cell or animal model is combined by the expression vector物 Preparation obtained.
更优选地，所述细胞或动物模型为含有MBP基因大片段缺少的纯合子细胞或动物模型。More preferably, the cell or animal model is a homozygous cell or animal model containing a lack of a large fragment of the MBP gene.
本发明所述方法采用的是两条分别靶向猪MBP基因外显子1和外显子6的sgRNA序列，通过筛选(更佳为通过单次细胞筛选)即可获得大片段基因敲除细胞系，本方法操作简单，与传统的基因修饰方法如Cre-loxP(时间更短)，显微注射(无嵌合体)更加高效。通过此方法获得的猪MBP基因敲除细胞系可直接用于体细胞克隆获得基因修饰动物猪，猪由于在体型及模拟人类神经系统上相较于小鼠更有优势，因此在今后相关疾病的致病机制及疾病治疗方案研究方面发挥更大的作用。The method of the present invention adopts two sgRNA sequences respectively targeting exon 1 and exon 6 of pig MBP gene, and large fragment gene knockout cells can be obtained through screening (preferably through single cell screening) This method is simple to operate, and is more efficient than traditional gene modification methods such as Cre-loxP (shorter time) and microinjection (no chimerism). The pig MBP gene knockout cell line obtained by this method can be directly used for somatic cell cloning to obtain genetically modified animal pigs. Because pigs have more advantages than mice in terms of body size and simulation of human nervous system, they will be used in the future for related diseases. Play a greater role in the study of pathogenic mechanisms and disease treatment options.
附图说明Description of the drawings
图1为本发明方法构建敲除MBP基因的猪成纤维细胞Sanger测序结果。Figure 1 shows the results of Sanger sequencing of porcine fibroblasts with MBP gene knocked out constructed by the method of the present invention.
具体实施方式detailed description
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.
实施例1用于猪MBP基因敲除的sgRNA组合物及其表达载体Example 1 sgRNA composition and its expression vector for pig MBP gene knockout
1.Cas9靶位点的选择1. The choice of Cas9 target site
Cas9靶位点包含20个碱基，紧邻靶点3’端的3个碱基构成PAM区，PAM区要求序列为NGG(N为任意碱基)。根据该原则设计，针对猪MBP基因第1外显子和第6外显子分别设计一条sgRNA，从5’-3’方向序列如下：The Cas9 target site contains 20 bases, and the 3 bases immediately adjacent to the 3'end of the target constitute the PAM region. The PAM region requires a sequence of NGG (N is any base). Designed according to this principle, one sgRNA was designed for the first exon and the sixth exon of the pig MBP gene, and the sequence from the 5'-3' direction is as follows:
针对第1外显子设计的sgRNA序列：GAGGCACGGATCAAAGTACC(SEQ ID NO.1)；相应的正义链序列和反义链序列为：The sgRNA sequence designed for the first exon: GAGGCACGGATCAAAGTACC (SEQ ID NO.1); the corresponding sense strand sequence and antisense strand sequence are:
1F：
Figure PCTCN2020114926-appb-000001
(SEQ ID NO.3)， 1F:
Figure PCTCN2020114926-appb-000001
(SEQ ID NO.3),
1R：
Figure PCTCN2020114926-appb-000002
(SEQ ID NO.4)； 1R:
Figure PCTCN2020114926-appb-000002
(SEQ ID NO.4);
针对第6外显子设计的sgRNA序列：GAGGACGGGACAGCCGCTCC(SEQ ID NO.2)；相应的正义链序列和反义链序列为：The sgRNA sequence designed for exon 6: GAGGACGGGACAGCCGCTCC (SEQ ID NO. 2); the corresponding sense strand sequence and antisense strand sequence are:
2F：
Figure PCTCN2020114926-appb-000003
(SEQ ID NO.5)， 2F:
Figure PCTCN2020114926-appb-000003
(SEQ ID NO.5),
2R：
Figure PCTCN2020114926-appb-000004
(SEQ ID NO.6)； 2R:
Figure PCTCN2020114926-appb-000004
(SEQ ID NO.6);
加粗部分包括BbsI酶切位点。The bold part includes the BbsI restriction site.
2.CRISPR Cas9质粒构建2. CRISPR Cas9 plasmid construction
(1)酶切骨架质粒：骨架质粒为PX459 V2.0(addgene,62988)，酶切体系如下表1，37℃酶切5h左右，回收之前先取5μL产物跑胶确认酶切完全。(1) Enzyme digestion of backbone plasmid: The backbone plasmid is PX459 V2.0 (addgene,62988). The digestion system is as shown in Table 1. Digestion at 37°C for about 5 hours. Before recovery, take 5μL of the product and run the gel to confirm that the digestion is complete.
表1酶切体系Table 1 Enzyme digestion system
PX459 V2.0PX459 V2.0 3μg3μg
BbsI(NEB，R3539S)BbsI (NEB, R3539S) 2μL2μL
10×Buffer10×Buffer 10μL10μL
ddH 2O ddH 2 O 补至100μLMake up to 100μL
采用胶回收试剂盒(TIANGEN)回收酶切产物(约9162bp)。The digested product (about 9162 bp) was recovered using the gel recovery kit (TIANGEN).
(2)Oligos磷酸化和退火：按下表2分别配制退火体系。(2) Oligos phosphorylation and annealing: the annealing system was prepared separately according to Table 2.
表2退火体系Table 2 Annealing system
Oligo F(100μM)Oligo F(100μM) 1μL1μL
Oligo R(100μM)Oligo R(100μM) 1μL1μL
10×T4 Ligation Buffer(NEB)10×T4 Ligation Buffer(NEB) 1μL1μL
T4 PNK(NEB,M0201S)T4 PNK(NEB,M0201S) 0.5μL0.5μL
ddH 2O ddH 2 O 6.5μL6.5μL
退火程序为：37℃ 30min，95℃ 5min，接着以5℃/min的速度降温至25℃。The annealing procedure is: 37°C for 30 minutes, 95°C for 5 minutes, and then the temperature is reduced to 25°C at a rate of 5°C/min.
(3)连接：按下表3配制连接体系，16℃连接过夜。(3) Connection: Prepare the connection system as shown in Table 3, and connect overnight at 16°C.
表3连接体系Table 3 Connection system
步骤(1)的酶切回收的产物The product recovered by digestion of step (1) 50ng50ng
步骤(2)的oligos(稀释200倍)The oligos of step (2) (diluted 200 times) 1μL1μL
T4 Ligase(NEB)T4 Ligase(NEB) 0.5μL0.5μL
10×T4 Ligation Buffer(NEB)10×T4 Ligation Buffer(NEB) 1μL1μL
ddH 2O ddH 2 O 补至10μLMake up to 10μL
(4)转化连接产物，挑取单克隆进行测序(pLKO1.5，测序公司通用引物)，得到连有sgRNA的表达载体组。(4) Transform the ligation product, pick a single clone for sequencing (pLKO1.5, general primer of the sequencing company), to obtain an expression vector group connected with sgRNA.
实施例2猪MBP基因敲除细胞的筛选Example 2 Screening of pig MBP gene knockout cells
1.嘌呤霉素筛选浓度的确定1. Determination of Puromycin Screening Concentration
猪成纤维细胞生长至80％左右后以1:10传代；24h后，加入含不同浓度嘌呤霉素的新鲜培养基(0-2μg/mL内设置至少5个浓度梯度)。每日显微镜下观察细胞存活比例(嘌呤霉素的最佳作用时间一般在1-4天)，选用加药2-3天杀死所有细胞的最低筛选浓度作为筛选细胞时选用的嘌呤霉素浓度。本实验的嘌呤霉素筛选浓度为800ng/μL。After porcine fibroblasts grow to about 80%, they are passaged at 1:10; 24 hours later, fresh media containing different concentrations of puromycin are added (at least 5 concentration gradients are set within 0-2μg/mL). Observe the cell survival ratio under the microscope every day (the best action time of puromycin is generally 1-4 days), and select the lowest concentration of puromycin that kills all cells for 2-3 days as the concentration of puromycin used for cell selection . The screening concentration of puromycin in this experiment is 800ng/μL.
2.细胞的转染与筛选2. Transfection and screening of cells
(1)在含有10％FBS的DMEM培养基培养猪成纤维细胞，待细胞长至70-90％左右时以1:2比例进行传代，当传代的细胞长至70-90％左右时，可以用于细胞转染；(1) Cultivate porcine fibroblasts in DMEM medium containing 10% FBS. When the cells grow to about 70-90%, pass them at a ratio of 1:2. When the passaged cells grow to about 70-90%, you can For cell transfection;
(2)通过电转转入实施例1的表达载体组；(2) Transfer to the expression vector group of Example 1 by electrotransfer;
(3)转染24h后，根据细胞生长情况分板，将细胞消化后以一定比例分至10cm皿培养板上(一般是细胞均匀分至10cm板上后，在显微镜下观察，4×物镜下一个视野看到10-20个细胞左右最佳)；(3) After 24 hours of transfection, divide the plates according to the growth of the cells, and divide the cells into a 10cm dish culture plate in a certain proportion after digestion (usually after the cells are evenly divided into the 10cm plate, observe under the microscope, 4× objective lens It is best to see about 10-20 cells in one field of view);
(4)分板24h后，将培养基换成含0.8μg/mL嘌呤霉素、10％FBS的DMEM培养基(每天观察细胞，嘌呤霉素一般作用两天即可换回原培养基，若发现10cm板上的细胞过多，两天之后可以继续加入含嘌呤霉素的培养基)；(4) After splitting the plate for 24 hours, change the medium to DMEM medium containing 0.8μg/mL puromycin and 10% FBS (observe the cells every day. Puromycin can be changed back to the original medium after two days. It is found that there are too many cells on the 10cm plate, you can continue to add the medium containing puromycin after two days);
(5)细胞筛选后10～14天左右在显微镜下可看到细胞克隆，通过克隆环(康宁，3166-8)胰酶消化法挑取细胞克隆，将每个细胞克隆挑取至24孔板中，待24孔细胞克隆长至90％左右时，1：2传代，并取出部分细胞进行PCR，测序鉴定。(5) About 10 to 14 days after cell selection, cell clones can be seen under the microscope. The cell clones are picked by trypsinization of the cloning ring (Corning, 3166-8), and each cell clone is picked to a 24-well plate When the 24-well cell clone grows to about 90%, pass 1:2, and take out some cells for PCR and sequencing for identification.
鉴定所用的上游引物一般设计在sgRNA位点上游100-200bp处，下游引物一般设计在sgRNA位点下游100-200bp处。本次实验的鉴定引物为：The upstream primer used for identification is generally designed at 100-200bp upstream of the sgRNA site, and the downstream primer is generally designed at 100-200bp downstream of the sgRNA site. The identification primers in this experiment are:
FP1：GGGAGGGAGGACAACACCTTCA，FP1: GGGAGGGAGGGACAACACCTTCA,
RP1：GGGATGTCACTGTCTCCGAGGTA。RP1: GGGATGTCACTGTCTCCGAGGTA.
PCR扩增条件为：The PCR amplification conditions are:
Figure PCTCN2020114926-appb-000005
Figure PCTCN2020114926-appb-000005
3.结果：3. Results:
共获得76个细胞克隆，经测序鉴定，其中大片段基因敲除纯合子克隆有3个，效率为3.95％(3/76)，这3个克隆分别为43号、63号及73号，获得3号为野生型，Sanger测序如图1。A total of 76 cell clones were obtained. After sequencing, there were 3 large-segment knockout homozygous clones with an efficiency of 3.95% (3/76). These 3 clones were No. 43, No. 63, and No. 73, respectively. No. 3 is wild type, Sanger sequencing is shown in Figure 1.
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地说明，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。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those of ordinary skill in the art should understand The technical solution of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

  1. 用于猪MBP基因敲除的sgRNA组合物，其特征在于，所述组合物包括sgRNA1和sgRNA2；所述sgRNA1和sgRNA2的核苷酸序列从5’到3’方向如SEQ ID NO.1和SEQ ID NO.2所示。The sgRNA composition for pig MBP gene knockout, characterized in that the composition comprises sgRNA1 and sgRNA2; the nucleotide sequence of the sgRNA1 and sgRNA2 is from 5'to 3', such as SEQ ID NO.1 and SEQ ID NO.2.
  2. 如权利要求1所述的sgRNA组合物在构建敲除猪MBP基因的sgRNA表达载体组合物中的应用。The use of the sgRNA composition according to claim 1 in constructing a sgRNA expression vector composition for knocking out the pig MBP gene.
  3. 用于敲除猪MBP基因的sgRNA表达载体组合物，其特征在于，所述表达载体包括表达载体1和表达载体2；所述表达载体1含有如SEQ ID NO.1所示的序列，所述表达载体2含有如SEQ ID NO.2所示的序列。The sgRNA expression vector composition for knocking out the pig MBP gene is characterized in that the expression vector includes expression vector 1 and expression vector 2; the expression vector 1 contains the sequence shown in SEQ ID NO.1, and The expression vector 2 contains the sequence shown in SEQ ID NO.2.
  4. 如权利要求3所述的表达载体组合物，其特征在于，所述表达载体组合物的载体骨架为pX459。8. The expression vector composition of claim 3, wherein the vector backbone of the expression vector composition is pX459.
  5. 如权利要求3或4所述的表达载体组合物的构建方法，其特征在于，包括如下步骤：The method for constructing an expression vector composition according to claim 3 or 4, characterized in that it comprises the following steps:
    (1)合成如权利要求1所述sgRNA对应的正义链和反义链；(1) Synthesize the sense strand and antisense strand corresponding to the sgRNA of claim 1;
    (2)酶切载体骨架，并回收酶切产物；(2) Enzyme digestion of the vector backbone, and recovery of the digestion product;
    (3)分别将步骤(1)所述sgRNA对应的正义链和反义链进行退火得到双链；(3) Annealing the sense strand and antisense strand corresponding to the sgRNA in step (1) respectively to obtain a double strand;
    (4)将步骤(3)的双链分别连接到步骤(2)的酶切产物上，转化鉴定，即得所述表达载体组合物。(4) Connect the double strands of step (3) to the digested products of step (2) respectively, and transform and identify them to obtain the expression vector composition.
  6. 如权利要求1所述的sgRNA组合物、如权利要求3或4所述表达载体组合物在制备MBP基因敲除的细胞系或动物模型中的用途。Use of the sgRNA composition according to claim 1 and the expression vector composition according to claim 3 or 4 in the preparation of MBP gene knockout cell lines or animal models.
  7. 制备MBP基因敲除细胞的方法，其特征在于，包括将如权利要求3或4所述的表达载体组合物转入细胞中。The method for preparing MBP gene knockout cells is characterized in that it comprises transferring the expression vector composition according to claim 3 or 4 into the cells.
  8. 如权利要求7所述的方法制备的MBP基因敲除细胞。The MBP gene knockout cell prepared by the method of claim 7.
  9. 如权利要求8所述的细胞，其特征在于，所述细胞为猪成纤维细胞。The cell of claim 8, wherein the cell is a porcine fibroblast.
  10. 一种MBP基因敲除的细胞或动物模型在用于治疗MBP基因缺陷引起的疾病的药物筛选中的用途，其特征在于，所述MBP基因敲除的细胞或动物模型通过如权利要求3或4所述表达载体组合物制备得到。The use of an MBP gene knockout cell or animal model in drug screening for the treatment of diseases caused by MBP gene defects, characterized in that the MBP gene knockout cell or animal model is as claimed in claim 3 or 4. The expression vector composition is prepared.
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CN114934068A (en) * 2022-06-24 2022-08-23 浙江大学医学院附属妇产科医院 Mouse ovary granular cell strain with stable ALKBH5 gene knockout function, construction method and application

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438083A (en) * 2022-01-29 2022-05-06 五邑大学 sgRNA for identifying pig PERV gene and coding DNA and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821049A (en) * 2016-04-29 2016-08-03 中国农业大学 Production method for Fbxo40 gene knockout pigs
WO2018110805A1 (en) * 2016-12-16 2018-06-21 주식회사 엠젠플러스 Animal model of diabetes with ins gene knockout or diabetic complications and manufacturing method thereof
CN108221058A (en) * 2017-12-29 2018-06-29 苏州金唯智生物科技有限公司 One boar full-length genome sgRNA libraries and its construction method and application
CN110607280A (en) * 2019-08-28 2019-12-24 华中农业大学 Application of EMC3 gene and site-directed knockout method thereof
CN111172191A (en) * 2020-02-21 2020-05-19 浙江大学 Efficient gene knockout vector and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003079270A (en) * 2001-09-10 2003-03-18 Japan Science & Technology Corp Myelin development inhibitory non-human animal model
US7423194B2 (en) * 2005-06-14 2008-09-09 University Of Chicago Animal models for demyelination disorders
JP2013046597A (en) * 2011-07-26 2013-03-07 Osaka Univ Model animal and cell for nervous system disease and their use
CN109096403B (en) * 2018-07-04 2020-06-30 浙江工商大学 Protein vector for protein transduction and preparation method and application thereof
KR20210105886A (en) * 2018-11-08 2021-08-27 고쿠리츠 다이가쿠 호우징 도우카이 고쿠리츠 다이가쿠 기코우 Gene therapy using genome editing with a single AAV vector

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821049A (en) * 2016-04-29 2016-08-03 中国农业大学 Production method for Fbxo40 gene knockout pigs
WO2018110805A1 (en) * 2016-12-16 2018-06-21 주식회사 엠젠플러스 Animal model of diabetes with ins gene knockout or diabetic complications and manufacturing method thereof
CN108221058A (en) * 2017-12-29 2018-06-29 苏州金唯智生物科技有限公司 One boar full-length genome sgRNA libraries and its construction method and application
CN110607280A (en) * 2019-08-28 2019-12-24 华中农业大学 Application of EMC3 gene and site-directed knockout method thereof
CN111172191A (en) * 2020-02-21 2020-05-19 浙江大学 Efficient gene knockout vector and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, XIAOXUE ET AL.: "Establishment of Bama Miniature Pig PFFS with GGTA1/β4GalNT2 Deficiency by Double Gene Knockout", PRACTICAL JOURNAL OF ORGAN TRANSPLANTATION (ELECTRONIC VERSION), vol. 7, no. 4, 31 July 2019 (2019-07-31), pages 277 - 282, XP055876171, ISSN: 2095-5332 *
PAPAGEORGIOU K. V., GRIVAS I., CHIOTELLI M., PANTERIS E., PAPAIOANNOU N., NAUWYNCK H., KRITAS S. K.: "Myelin Sheath Development in the Maxillary Nerve of the Newborn Pig", ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, vol. 46, no. 1, 1 February 2017 (2017-02-01), DE , pages 58 - 64, XP055876178, ISSN: 0340-2096, DOI: 10.1111/ahe.12234 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114934068A (en) * 2022-06-24 2022-08-23 浙江大学医学院附属妇产科医院 Mouse ovary granular cell strain with stable ALKBH5 gene knockout function, construction method and application
CN114934068B (en) * 2022-06-24 2024-04-19 浙江大学医学院附属妇产科医院 Mice ovary granule cell strain with stable ALKBH5 gene knockout, construction method and application

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