WO2020000465A1 - Procédé de préparation de souris à invalidation du gène lightr - Google Patents

Procédé de préparation de souris à invalidation du gène lightr Download PDF

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Publication number
WO2020000465A1
WO2020000465A1 PCT/CN2018/093875 CN2018093875W WO2020000465A1 WO 2020000465 A1 WO2020000465 A1 WO 2020000465A1 CN 2018093875 W CN2018093875 W CN 2018093875W WO 2020000465 A1 WO2020000465 A1 WO 2020000465A1
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WIPO (PCT)
Prior art keywords
lightr
gene
crispr
sgrna
cell
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PCT/CN2018/093875
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English (en)
Chinese (zh)
Inventor
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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Priority to PCT/CN2018/093875 priority Critical patent/WO2020000465A1/fr
Publication of WO2020000465A1 publication Critical patent/WO2020000465A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the invention relates to the fields of genetic engineering and gene editing, and in particular, to editing a LIGHTR gene by using a CRISPR-Cas9 system and obtaining a LIGHTR gene knockout mouse through a somatic cell nuclear transfer technology.
  • LIGHTR belongs to the tumor necrosis factor receptor (TNFR) family of co-stimulatory molecules. Studies have shown that LIGHTR can interact with the CD28 family of co-stimulatory molecular members-B and T lymphocyte attenuation factors and inhibit T cell proliferation and cytokine production, suggesting that LIGHTR-centered immune regulatory signals play a key role in the immune response.
  • TNFR tumor necrosis factor receptor
  • LIGHTR can play an important role in the treatment of autoimmune diseases, tumors and other diseases.
  • a large amount of research is required to achieve clinical transformation.
  • a vector expressing the LIGHTR gene and a cell line with high expression of the LIGHTR gene are lacking. Research progress has caused certain obstacles.
  • 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 for the research of LIGHTR gene function.
  • an object of the present invention is to provide a method for preparing a LIGHTR gene knockout mouse.
  • the present invention first provides an sgRNA that specifically targets the LIGHTR gene, the nucleotide sequence of which is 5 '-CAGGTTGGGAGGCTACAGGA-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. The oligo sequence is annealed by boiling for 5 minutes, then left to 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 LIGHTR gene knockout cell, which transfects a CRISPR-Cas9 targeting vector containing a sgRNA targeted to the CDS region of the LIGHTR gene, thereby knocking out the LIGHTR gene of the cell.
  • the present invention also provides a method for preparing a LIGHTR gene knockout mouse, that is, the LIGHTR gene is knocked out by using a CRISPR-Cas9 targeting vector that targets the CDS region of the LIGHTR gene.
  • 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 LIGHTR gene for the first time is cut by the CRISPR-Cas9 system LIGHTR gene to obtain a LIGHTR knockout mouse individual.
  • This method for preparing a LIGHTR knockout mouse is domestically Was not reported before. It provides a practical method for studying mouse LIGHTR 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 LIGHTR gene, and its sequence is 5 ’- CAGGTTGGGAGGCTACAGGA -3 ', according to the principle of base complementary pairing, its reverse complementary sequence is 5'- TCCTGTAGCCTCCCAACCTG -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 LIGHTR 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. Cell culture medium to terminate digestion, 1000 Centrifuge at rpm for 5 min, discard the supernatant, resuspend the centrifuged cells, and place 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 LIGHTR gene for the first time is cut by the CRISPR-Cas9 system LIGHTR gene to obtain a LIGHTR knockout mouse individual.
  • This method for preparing a LIGHTR knockout mouse is domestically Was not reported before. It provides a practical method for studying mouse LIGHTR gene.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne l'utilisation du système CRISPR-Cas9 pour modifier le gène LIGHTR, et les souris à invalidation du gène LIGHTR qui sont obtenues au moyen d'une technologie de transfert nucléaire de cellules somatiques. L'ARNsg est conçu pour la première fois dans la région CDS du gène LIGHTR de souris, et celui-ci est coupé à l'aide du système CRISPR-Cas9, ce qui permet d'obtenir une invalidation du gène LIGHTR et d'obtenir un individu de souris à invalidation du gène correspondant ; un procédé pratique d'étude du gène LIGHTR de souris est ainsi fourni.
PCT/CN2018/093875 2018-06-29 2018-06-29 Procédé de préparation de souris à invalidation du gène lightr WO2020000465A1 (fr)

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PCT/CN2018/093875 WO2020000465A1 (fr) 2018-06-29 2018-06-29 Procédé de préparation de souris à invalidation du gène lightr

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PCT/CN2018/093875 WO2020000465A1 (fr) 2018-06-29 2018-06-29 Procédé de préparation de souris à invalidation du gène lightr

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WO2020000465A1 true WO2020000465A1 (fr) 2020-01-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016106295A1 (fr) * 2014-12-22 2016-06-30 Dana-Farber Cancer Institute, Inc. Méthodes in vivo permettant d'identifier des cibles d'immunothérapie associées au cancer
CN106163547A (zh) * 2014-03-15 2016-11-23 诺华股份有限公司 使用嵌合抗原受体治疗癌症
WO2018064602A1 (fr) * 2016-09-30 2018-04-05 Baylor College Of Medicine Thérapie par récepteurs d'antigène chimériques présentant une cytotoxicité réduite pour maladie virale

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106163547A (zh) * 2014-03-15 2016-11-23 诺华股份有限公司 使用嵌合抗原受体治疗癌症
WO2016106295A1 (fr) * 2014-12-22 2016-06-30 Dana-Farber Cancer Institute, Inc. Méthodes in vivo permettant d'identifier des cibles d'immunothérapie associées au cancer
WO2018064602A1 (fr) * 2016-09-30 2018-04-05 Baylor College Of Medicine Thérapie par récepteurs d'antigène chimériques présentant une cytotoxicité réduite pour maladie virale

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