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CN-122012690-A - Gene knockout mouse model construction method based on ichthyosis pathogenic gene GLTP and application

CN122012690ACN 122012690 ACN122012690 ACN 122012690ACN-122012690-A

Abstract

The invention discloses a gene knockout mouse model construction method based on ichthyosis pathogenic gene GLTP and application thereof. The NCBI gene ID of the GLTP gene is 51228. The Gltp gene knockout mouse model is constructed by assembling sgRNA of a target mouse Gltp gene and Cas9 protein in vitro, performing CRISPR/Cas9-sgRNA compound, microinjecting the compound into a mouse fertilized egg, transplanting the fertilized egg to obtain a positive F0 generation mouse, mating the positive F0 generation mouse with a wild type mouse, and screening heterozygotes, and selfing the heterozygotes. The invention definitely identifies GLTP gene as a new pathogenic gene of ichthyosis for the first time, and the established Gltp gene knockout mouse model can spontaneously generate ichthyosis-like skin lesions, simulate human disease characteristics and provide a more ideal tool for researching pathogenesis of ichthyosis and screening targeted drugs.

Inventors

  • WANG HUIJUN
  • LIN ZHIMIAO
  • ZHANG ZEQIAO

Assignees

  • 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心）

Dates

Publication Date
20260512
Application Date
20260129

Claims (10)

  1. 1. Application of a reagent for detecting GLTP gene mutation in preparing a kit for diagnosing or assisting in diagnosing ichthyosis, wherein NCBI gene ID of the GLTP gene is 51228.
  2. 2. The use according to claim 1, wherein the reagent is a primer or a probe.
  3. 3. The use according to claim 1, wherein the site of mutation comprises one or two of c.49c > T, c.58_62del, c.85delc, c.98delt and c.180c > a; And/or, the reagent comprises: (a) The primer pair for detecting c.49C > T, c.58_62del, c.85delC and c.98delT has a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 2; (b) The nucleotide sequence of the primer pair for detecting c.180C > A is shown as SEQ ID NO. 5-SEQ ID NO. 6.
  4. 4. A kit for diagnosis or auxiliary diagnosis of ichthyosis, comprising a reagent for detecting mutation of GLTP gene, wherein NCBI gene ID of the GLTP gene is 51228.
  5. 5. The kit of claim 4, wherein the site of mutation comprises one or two of c.49c > T, c.58_62del, c.85delc, c.98delt, and c.180c > a; And/or, the reagent comprises: (a) The primer pair for detecting c.49C > T, c.58_62del, c.85delC and c.98delT has a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 2; (b) The nucleotide sequence of the primer pair for detecting c.180C > A is shown as SEQ ID NO. 5-SEQ ID NO. 6.
  6. 6. The construction method of Gltp gene knockout mouse model is characterized by comprising the following steps: (1) Incubating and assembling sgRNA of the targeted mouse Gltp gene and Cas9 protein in vitro to obtain a CRISPR/Cas9-sgRNA complex; (2) Microinjection of the CRISPR/Cas9-sgRNA complex in the step (1) into fertilized eggs of mice, and transplantation of fertilized eggs to obtain positive F0-generation mice; (3) After mating the positive F0 generation mice with wild mice, screening heterozygotes, and selfing the heterozygotes to obtain Gltp gene knockout mouse models.
  7. 7. The method of claim 6, wherein the sgRNA in step (1) has the sequence shown in SEQ ID NO. 12 and SEQ ID NO. 13.
  8. 8. The method for constructing a Gltp gene knockout mouse model according to claim 6, wherein the Gltp gene knockout mouse model in the step (3) is characterized by comprising the steps of performing PCR amplification twice by using tissue DNA of a mouse tail tip as a template and using SEQ ID NO. 14-SEQ ID NO. 15 and SEQ ID NO. 16-SEQ ID NO. 17 as primers respectively, and performing electrophoretic analysis on amplified products; Preferably, the reaction system for PCR amplification comprises 2X PHANTA FLASH MASTER Mix 12.5 mu L, ddH 2 O9.5 mu L, forward primer and reverse primer of 1 mu L each and template of 1 mu L, wherein the reaction process for PCR amplification comprises the steps of 98 ℃ pre-denaturation for 30 seconds, then 35 cycles of 98 ℃ denaturation for 10 seconds, 62 ℃ annealing for 5 seconds, 72 ℃ extension for 5 seconds and finally 72 ℃ final extension for 1 minute; Preferably, if the primer of SEQ ID NO. 14-SEQ ID NO. 15 is used for PCR amplification, 382 bp bands appear in the amplified product, and the primer of SEQ ID NO. 16-SEQ ID NO. 17 is used for PCR amplification, NO band appears in the amplified product, namely the Gltp gene knockout mouse model.
  9. 9. The Gltp gene knockout mouse model constructed by the construction method according to any one of claims 6 to 8.
  10. 10. Use of the Gltp gene knockout mouse model of claim 9 in ichthyosis pathological mechanism research and/or screening of drugs for treating ichthyosis.

Description

Gene knockout mouse model construction method based on ichthyosis pathogenic gene GLTP and application Technical Field The invention belongs to the technical field of medical genetics, and particularly relates to identification of a new pathogenic gene GLTP of ichthyosis, a method for constructing Gltp gene knockout mouse model based on mouse Gltp genes and application thereof. Background Hereditary ichthyosis is a group of skin disorders characterized by abnormal skin keratinization, scaling and barrier dysfunction. Although part of the pathogenic genes and pathogenesis of ichthyosis have been elucidated initially in recent years, a significant proportion of patients have unknown pathogenic genes and their pathogenesis is unknown. Therefore, a new ichthyosis pathogenic gene is searched, the functional mechanism of the ichthyosis pathogenic gene is researched, the method has important significance for deeply understanding a gene regulation network of skin barriers, and a new drug target can be provided for clinical treatment. Disclosure of Invention Based on the above, the invention aims to provide a novel ichthyosis pathogenic gene, and a construction method and application of a gene knockout mouse model based on the pathogenic gene. The specific technical scheme for achieving the aim of the invention comprises the following steps. In a first aspect of the invention, there is provided the use of a reagent for detecting mutations in the GLTP gene having NCBI gene ID 51228 in the preparation of a kit for diagnosis or assisted diagnosis of ichthyosis. In a second aspect of the present invention, there is provided a kit for diagnosis or auxiliary diagnosis of ichthyosis comprising reagents for detecting mutation of GLTP gene having NCBI gene ID 51228. In a third aspect of the present invention, there is provided a method for constructing a Gltp gene knockout mouse model, comprising the steps of: (1) Incubating and assembling sgRNA of the targeted mouse Gltp gene and Cas9 protein in vitro to obtain a CRISPR/Cas9-sgRNA complex; (2) Microinjection of the CRISPR/Cas9-sgRNA complex in the step (1) into fertilized eggs of mice, and transplantation of fertilized eggs to obtain positive F0-generation mice; (3) After mating the positive F0 generation mice with wild mice, screening heterozygotes, and selfing the heterozygotes to obtain Gltp gene knockout mouse models. In a fourth aspect of the present invention, a Gltp gene knockout mouse model constructed by the above construction method is provided. In a fifth aspect of the present invention, there is provided the use of the Gltp gene knockout mouse model described above in ichthyosis pathological mechanism research and/or screening of drugs for treating ichthyosis. The inventor of the present invention found when 7 genetic ichthyosis patients were studied that the double allele frameshift or nonsense mutation (c.58_62 del, c.85delC, c.98delT, c.49C > T and c.180C > A) of the GLTP gene can cause the deficiency of GLTP gene expression, thereby causing ichthyosis, and the GLTP gene is definitely identified for the first time as a novel pathogenic gene of ichthyosis, and the complete molecular pathway of GLTP deficiency leading to skin barrier dysfunction through causing autophagy abnormality is revealed for the first time, thereby establishing a disease occurrence mechanism model of 'gene defect-autophagy block-barrier destruction'. The invention has important value in the aspect of transformation medical research, and can provide support for preclinical evaluation of gene therapy strategies, optimization of drug delivery systems and development of personalized treatment schemes. According to the invention, a gene knockout technology is utilized, the 2-4 exons of the Gltp gene of the mouse are selected as a targeting region, protein expression deletion after knockout is ensured, a Gltp gene knockout mouse model is successfully constructed, the mouse model can spontaneously generate ichthyosis-like skin lesions, human disease characteristics are simulated, and a more ideal tool is provided for researching ichthyosis disease pathogenesis and screening targeting drugs (small molecule drugs targeting GLTP channels, autophagy regulators and barrier function repair drugs). Drawings Fig. 1 is a diagram showing clinical manifestations of congenital ichthyosis patients. Fig. 2 is a diagram showing the pathological manifestations of skin lesions in congenital ichthyosis patients. FIG. 3 is a GLTP gene mutation sequencing map of congenital ichthyosis patients. Fig. 4 shows the results of skin GLTP immunofluorescence for normal control and congenital ichthyosis patients. Fig. 5 shows the results of the skin electron microscopy of normal control and congenital ichthyosis patients. FIG. 6 shows the results of autophagy index immunofluorescence of normal control and congenital ichthyosis patients. FIG. 7 shows Western Blot and immunofluorescence results of Gltp +/+ and Gltp -/- mouse