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CN-121975866-A - Construction method and application of transgenic mouse model for in-vivo autoimmune gene screening

CN121975866ACN 121975866 ACN121975866 ACN 121975866ACN-121975866-A

Abstract

The invention discloses a construction method and application of a transgenic mouse model for in-vivo autoimmune gene screening, belongs to the technical field of animal model construction, and aims to solve the problems of unrealistic in-vitro screening targets, poor repeatability, complex operation, narrow application range and lack of in-vivo screening tools for multiple autoimmune genes. The invention designs the sgRNA (SEQ ID NO: 1-150) of 140 autoimmune genes and 10 control genes, builds 150mer library plasmids step by step, mates with Cas9 and UBC-Cre ERT2 mice after microinjection to obtain three-positive mice, and combines a tumor model, flow sorting and NGS analysis screening targets to screen autoimmune related genes, can cover 150 autoimmune related genes, has the advantages of high screening reality, high repeatability, simple operation and wide application, and provides a key tool for autoimmune research and target development.

Inventors

  • LIU BO
  • SUN XIUFENG
  • ZHAO JINDONG
  • ZHAO ZIHAO
  • YANG YIJIE

Assignees

  • 合肥综合性国家科学中心大健康研究院
  • 安徽中医药大学第一附属医院(安徽省中医院)

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. The sgRNA library plasmid for targeting autoimmune genes is characterized by comprising sgRNA sequences aiming at 140 core autoimmune target genes and sgRNA sequences of 10 negative control genes, wherein the sgRNA sequences are shown as SEQ ID NO. 1-SEQ ID NO. 150.
  2. 2. The sgRNA library plasmid of claim 1, wherein the sgRNA sequence of the core autoimmune target gene is shown in SEQ ID NOs 1-9, 11-24, 26-39, 41-58, 61-74, 76-89, 91-104, 106-119, 121-134, 136-150, and the sgRNA sequence of the negative control gene is shown in SEQ ID NOs 10, 25, 40, 59, 60, 75, 90, 105, 120, 135.
  3. 3. The method for constructing the sgRNA library plasmid according to claim 1, wherein the method comprises the steps of assembling the sequences shown in SEQ ID NO. 1-15 into a 15mer-1 plasmid, assembling the sequences shown in SEQ ID NO. 16-30 into a 15mer-2 plasmid, assembling the sequences shown in SEQ ID NO. 31-45 into a 15mer-3 plasmid, assembling the sequences shown in SEQ ID NO. 46-60 into a 15mer-4 plasmid, assembling the sequences shown in SEQ ID NO. 61-75 into a 15mer-5 plasmid, assembling the sequences shown in SEQ ID NO. 76-90 into a 15mer-6 plasmid, assembling the sequences shown in SEQ ID NO. 91-105 into a 15mer-7 plasmid, assembling the sequences shown in SEQ ID NO. 106-120 into a 15mer-8 plasmid, assembling the sequences shown in SEQ ID NO. 121-135 into a 15mer-9, assembling the sequences shown in SEQ ID NO. 136-150 into a 15-10 plasmid, and assembling the 15mer-1 to 15mer-10 plasmid and the sgRNA library plasmid.
  4. 4. A method for constructing a transgenic mouse model for in vivo screening of autoimmune genes, comprising the steps of: S1, introducing the sgRNA library plasmid of claim 1 into a fertilized egg of a mouse through embryo microinjection, and transplanting the fertilized egg to a fallopian tube of a pseudopregnant female mouse to obtain an F0 generation mouse; S2, hybridizing the F0 generation positive mice with Ubc-Cre ERT2 and CAG-Cas9 double-positive tool mice, and screening to obtain three positive mice, namely the transgenic mouse model.
  5. 5. The method of claim 4, wherein the 150mer library plasmid is integrated into the mouse chromosome 8 at a position between positions 136395275 and 136395276 by microinjection.
  6. 6. The method of claim 4, wherein the transgenic mouse model is a positive mouse carrying three target elements of Ubc-Cre ERT2 gene, cas9 gene and 150mer library simultaneously.
  7. 7. Use of the transgenic mouse model prepared by the construction method of any one of claims 4-6 for in vivo screening of autoimmune genes.
  8. 8. The application according to claim 7, characterized in that said application comprises the steps of: (1) Inducing the mouse model to enable target cells to start sgRNA expression; (2) Inoculating a tumor cell construct in vivo screening environment; (3) Collecting a sample after the screening period is over; (4) Extracting sample genome DNA and constructing an NGS library; (5) Sequencing analysis of the relative abundance of each sgRNA, screening was performed based on the relative abundance of each sgRNA.
  9. 9. The use according to claim 8, wherein the sample in step (3) comprises any one or more of tumor tissue, spleen, small intestine, lung, stomach, esophagus, liver, kidney, colon, muscle, white fat, brown fat, lymph node, sexual organ, brain, cerebellum, thyroid, bone marrow, and thymus.
  10. 10. The use according to claim 8, wherein the high relative abundance of the sgrnas in step (5) indicates that the corresponding cells have survival or proliferation advantages in tumor microenvironments or in vivo screening systems after the sgrnas are knocked out, and that the loss of function of the autoimmune genes does not inhibit cell survival or even promote cell adaptation to the screening environment; the low relative abundance of the sgrnas indicates that the survival or proliferation of the corresponding cells in the in vivo screening system is significantly inhibited after the targeted autoimmune genes of the sgrnas are knocked out, indicating that the autoimmune genes are necessary to maintain the survival, proliferation or function of the cells in the screening environment.

Description

Construction method and application of transgenic mouse model for in-vivo autoimmune gene screening Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to a construction method and application of a transgenic mouse model for in-vivo autoimmune gene screening. Background Autoimmune diseases are pathological conditions that the immune system erroneously recognizes and attacks the healthy tissues and cells of itself, including various types of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus, autoimmune hepatitis, and the like, affecting the health of millions of people. The clinical manifestations of the diseases are complex, from slight symptoms such as fatigue and arthralgia to serious consequences such as organ failure and vision loss, the quality of life of patients is obviously reduced, high medical cost and social burden are brought, and most diseases are lack of radical treatment means at present. ("decryption of autoimmune diseases Using genomics" (Illumina because of the Mena, industry technical reviews), "ADVANCES IN CRISPR/CAS GENE THERAPY for inborn errors of immunity" (PMC, international journal reviews), "screening method for highly contributing pathogenic genes to rheumatoid arthritis The pathogenesis of autoimmune diseases is closely related to the synergism of genetic factors and environmental factors, wherein the mutation or abnormal expression of autoimmune genes is a core genetic predisposition. Autoimmune genes are not specific to a certain gene, but rather are a class of genes involved in the processes of immune regulation, inflammatory response, apoptosis, etc., whose dysfunction can destroy the recognition balance of the immune system "self-non-self" and trigger abnormal immune attack. Functional research and target screening of autoimmune genes are key to understanding pathogenesis of autoimmune diseases and developing related therapeutic drugs, however, the current screening technology aiming at autoimmune genes still has a plurality of defects, and the promotion and clinical transformation of related researches are severely restricted. At present, screening of autoimmune genes mainly depends on in-vitro screening technology, but the technology has a plurality of inherent defects that an in-vitro screening system cannot truly simulate complex physiological microenvironments in vivo, key factors such as cell interaction, tissue specificity regulation signals, immune microenvironment and the like often cause target spot screening omission or false positive results, meanwhile, the in-vitro screening technology is extremely poor in repeatability, tiny differences under different experimental conditions can cause significant deviation of screening results, the operation flow is complicated, cells and packaging viruses need to be prepared again for each screening, time and labor are consumed, in addition, the application range of the in-vitro screening technology is limited, and the delivery of gene editing tools cannot be realized due to insensitivity to virus infection of cells derived from a plurality of tissues, so that relevant gene screening cannot be carried out. ("A Fully Optimized CRISPR Workflow for Drug Discovery In T Cells" (Maxcyte, technical scheme report ）"AB0063 High-efficiency transduction of mesenchymal stem cells by aav2/dj vector"（Annals of the Rheumatic Diseases, International journal paper), "In vivo CAR-T technical bottleneck depth analysis" (EurekaBio, report on Industrial technical analysis)) CRISPR/Cas9 mediated in vivo gene screening technology provides possibility for solving the defects of the in vitro screening technology, but the application of the technology in the field of autoimmune gene screening still has obvious bottleneck at present. Specifically, there is no transgenic mouse model specially used for in vivo autoimmune gene screening at present, and the existing in vivo CRISPR screening technology is only widely used for primary T cells, and has few successful cases in primary NK cells, and cannot meet the requirements of multiple tissues and multiple cell types for autoimmune gene development regulation and tumor immunity related screening. The related patent documents: Publication No. CN117165627A, publication No. 2023.12.05, discloses a nucleic acid construct based on Cre-LoxP recombination system and CRISPR gene editing system and application thereof. The Cre-LoxP recombination system comprises a Cre enzyme and a LoxP nucleic acid combination, wherein the LoxP nucleic acid combination comprises TATA-Lox71 and TATA-LoxTC sequences, the recombination can only occur once under the catalysis of the Cre enzyme, and the nucleic acid construct carries inert filling sequences with a certain length. The nucleic acid construct of the invention is capable of expressing multiple sgrnas with low bias in vivo, but the same cell can only express one, thereby efficiently producing genetic chim