Search

CN-121992042-A - Construction method and application of zebra fish skeletal muscle defect model

CN121992042ACN 121992042 ACN121992042 ACN 121992042ACN-121992042-A

Abstract

The invention belongs to the field of disease model construction, and particularly relates to a construction method and application of a zebra fish skeletal muscle defect model. The method comprises the step of specifically knocking out Fgf8a gene in zebra fish based on CRISPR/Cas9 technology, wherein the target site sequence of gRNA is shown as SEQ ID NO. 1. The zebra fish skeletal muscle defect model constructed by the invention has obvious defect on rapid muscle fiber development, serious body axis bending condition, shortened dorsum abdomen wheelbase and left-right asymmetry. The model can be used for researching molecular mechanism of diseases and screening medicines for treating skeletal muscle diseases.

Inventors

  • ZHANG MINHONG
  • LU HUIQIANG
  • HUANG YONG
  • HUANG YUSHAN
  • Shen Tianzhu
  • FAN GUOQIANG

Assignees

  • 赣南医科大学第一附属医院

Dates

Publication Date
20260508
Application Date
20260306

Claims (8)

  1. 1. The construction method of the zebra fish skeletal muscle defect model is characterized by comprising the following steps of: injecting gRNA of Fgf8a gene of knockout zebra fish and Cas9 protein into embryo of wild zebra fish in a cell stage, incubating the embryo after fertilization for 2-4 days, observing embryo phenotype, screening embryo which normally develops for sequencing, screening mutant F0 generation, and further homozygous mutation screening to obtain the zebra fish with Fgf8a gene knockout, namely the zebra fish skeletal muscle defect model, wherein the target site sequence of the gRNA is shown as SEQ ID NO. 1.
  2. 2. The method for constructing a zebra fish skeletal muscle defect model according to claim 1, wherein the gRNA for knocking out zebra Fgf8a gene is prepared by the following steps: S1, performing PCR amplification on a gRNA template by using a primer shown in SEQ ID NO. 2-3 to obtain a double-stranded DNA template required by synthesis of gRNA, wherein the gRNA template is obtained by cloning Cas9 cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease; S2, performing in-vitro transcription on the DNA template to prepare gRNA; S3, purifying the prepared gRNA to obtain pure gRNA.
  3. 3. The method for constructing a zebra fish skeletal muscle defect model according to claim 2, wherein in S1, a PCR amplification system comprises 1 μl of a gRNA template, 1 μl of each of primers shown in SEQ ID NO. 2-3, 4 μl of a dNTP mixture, 5x Primer star buffer 10 μl of a PRIMER STAR enzyme, 1 μl of ddH 2 O32 μl.
  4. 4. The method according to claim 3, wherein in S1, the PCR amplification is performed for a total of 35 cycles at 98℃for 2 min, at 98℃for 15S, at 58℃for 15S, at 72℃for 20S, and at 72℃for 5min, at 30 min.
  5. 5. The method for constructing a skeletal muscle defect model of zebra fish according to claim 4, wherein in S2, the transcription system comprises 10. Mu.l of template DNA, 2. Mu.l of 10 XRNA polymerase reaction buffer, 0.8. Mu.l of 25 mM rNTP mixture, 0.5. Mu.l of RNase inhibitor, 2. Mu.l of T7 RNA polymerase and 4.7. Mu.l of RNase-free ddH 2 O.
  6. 6. The method according to claim 5, wherein in S2, the transcription procedure is 37 ℃ and 3 h.
  7. 7. The method for constructing a zebra fish skeletal muscle defect model according to claim 1, wherein the sequencing primer sequences are shown in SEQ ID NO. 4-5.
  8. 8. Use of the zebra fish skeletal muscle defect model constructed by the construction method of any one of claims 1 to 7 in screening a skeletal muscle disease treatment drug.

Description

Construction method and application of zebra fish skeletal muscle defect model Technical Field The invention belongs to the field of disease model construction, and particularly relates to a construction method and application of a zebra fish skeletal muscle defect model. Background Skeletal muscle accounts for about 50% of the total mass of the human body, and is a core organ for maintaining the motor function of the body. Its normal growth and development is an important indicator of physical health, whereas dysplasia can lead to skeletal muscle disease (myopathy). According to the statistics of world health organization, about 17.1 hundred million people of myopathy patients cover all ages from newborns to old people, and the trend is rising with the aging of population. Myopathy not only seriously threatens human health and life quality, but also brings heavy burden to individuals, families and society. Therefore, the molecular regulation mechanism of skeletal muscle development is studied deeply, the mechanism of dysplasia pathogenesis is clarified, and the method has important clinical and scientific values for diagnosing and treating myopathy. Skeletal muscle development undergoes key stages such as fate decisions, proliferation and differentiation of muscle precursor cells, and morphogenesis of muscle fibers, ultimately forming two main types, fast and slow muscle fibers. The existing research shows that the Hedgehog signal pathway is critical for the formation of slow muscle fibers-the ligand deficiency thereof can lead to significant reduction or even deletion of slow muscle fibers, but the regulation effect of the signal on fast muscle fibers is limited. In contrast, the molecular mechanism of fast muscle fiber development has not been clarified so far. The fibroblast growth factor (Fibroblast Growth Factor, fgf) family plays a key role in regulating cell proliferation, growth and differentiation, and abnormal expression of its members (e.g., fgf11, fgf16, fgf18 and receptors Fgfr1c, fgfr2c, fgfr4, etc.) is closely related to muscular dystrophy. Among them, fgf8, an important member of this family, has been demonstrated to have a function of regulating morphogenesis in embryonic heart, craniofacial and limb development. Studies have shown initially that fcf 8 is expressed in the tail buds and somites of vertebrate embryos, but its physiological function in somites and specific mechanisms of action in fast muscle development remain lacking in systematic studies. Disclosure of Invention In view of the limitations of the prior art, the invention provides a construction method and application of a zebra fish skeletal muscle defect model. The invention adopts the following technical scheme: in one aspect, the invention provides a method for constructing a zebra fish skeletal muscle defect model, which comprises the following steps: injecting gRNA of Fgf8a gene of knockout zebra fish and Cas9 protein into embryo of wild zebra fish in a cell stage, incubating the embryo after fertilization for 2-4 days, observing embryo phenotype, screening embryo which normally develops for sequencing, screening mutant F0 generation, and further homozygous mutation screening to obtain the zebra fish with Fgf8a gene knockout, namely the zebra fish skeletal muscle defect model, wherein the target site sequence of the gRNA is shown as SEQ ID NO. 1. The invention successfully constructs fgf a gene deletion mutant zebra fish. The research shows that the mutant shows typical skeletal muscle defect phenotype such as serious body axis bending, dorsiflexion wheelbase shortening, left-right asymmetry and the like in the embryo development process. The establishment of the model provides a key experimental tool and theoretical basis for deeply analyzing the molecular mechanism of congenital muscle diseases. Further, the gRNA of the knockout zebra fish Fgf8a gene is prepared by the following steps: S1, performing PCR amplification on a gRNA template by using a primer shown in SEQ ID NO. 2-3 to obtain a double-stranded DNA template required by synthesis of gRNA, wherein the gRNA template is obtained by cloning Cas9 cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease; S2, performing in-vitro transcription on the DNA template to prepare gRNA; S3, purifying the prepared gRNA to obtain pure gRNA. Further, in S1, the PCR amplification system was 1. Mu.l of the gRNA template, 1. Mu.l of each of the primers shown in SEQ ID NO.2 to 3, 4. Mu.l of dNTP mixture, 5x Primer star buffer 10. Mu.l, 1. Mu.l of PRIMER STAR enzyme, and 32. Mu.l of ddH 2 O. Further, in S1, the PCR amplification procedure was 98℃for 2 min, 98℃for 15S, 58℃for 15S, 72℃for 20S, 72℃for 5min, 30 min, for a total of 35 cycles. Further, in S2, the transcription system was 10. Mu.l of template DNA, 2. Mu.l of 10 XRNA polymerase reaction buffer, 0.8. Mu.l of 25 mM rNTP mixture, 0.5. Mu.l of RNase inhibitor, 2. Mu.l of T7 RNA polymerase and 4.7. Mu.l