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CN-122004174-A - Construction method and application of LgBiT transgenic mice

CN122004174ACN 122004174 ACN122004174 ACN 122004174ACN-122004174-A

Abstract

The invention discloses application of a NanoLuc luciferase double subunit complementary system in constructing an experimental animal model. The genome of the experimental animal model contains a NanoLuc enzyme large subunit LgBiT coding gene and stably expresses LgBiT, and a NanoLuc luciferase small subunit HiBiT polypeptide serving as a label or a NanoLuc luciferase small subunit HiBiT coding gene serving as a reporter gene is connected with a tested object which is used for invading the experimental animal model or is connected into the experimental animal model in a fusion way, and LgBiT and HiBiT are specifically combined in the experimental animal model to form the NanoLuc enzyme with catalytic activity in a recombination way. And a strong bioluminescence signal is generated under the action of a furazine substrate, so that high-sensitivity, non-invasive and real-time dynamic imaging of in-vivo distribution, loading capacity and diffusion paths of a tested object is realized. The invention also discloses a gene editing system for constructing LgBiT transgenic mice.

Inventors

  • XU JIANQING
  • ZHANG SHUYE
  • ZHANG XIAOYAN
  • SHU JIAYI
  • XIE XINGCHEN
  • LI XIAOHONG

Assignees

  • 复旦大学附属中山医院

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. Use of NanoLuc luciferase dual subunit complementary system for the construction of an experimental animal model for studying pathogen invasion of the body, pathogen proliferation or metabolism and extinction in the body, pathogen transfer and distribution in the body, infection and distribution of protein and nucleic acid drugs and vaccines in the body.
  2. 2. The use according to claim 1, wherein the genome of the experimental animal model comprises a gene encoding a large subunit LgBiT of a NanoLuc enzyme (the nucleotide sequence is shown as SEQ ID NO: 1), and the experimental animal model stably expresses LgBiT (the amino acid sequence is shown as SEQ ID NO: 2); Correspondingly, the tested object used for invading or being connected into the experimental animal model is in fusion connection with a NanoLuc luciferase small subunit HiBiT polypeptide (the amino acid sequence of which is shown as SEQ ID NO: 3) serving as a tag or a NanoLuc luciferase small subunit HiBiT coding gene (the nucleotide sequence of which is shown as SEQ ID NO: 4) serving as a reporter gene.
  3. 3. The use according to claim 1, wherein the animal is selected from the group consisting of monkey, pig, dog, sheep, rabbit, mouse, rat, zebra fish, drosophila, insect.
  4. 4. The method of claim 2, wherein the subject comprises a pathogen, a candidate protein drug, a candidate nucleic acid drug or a vaccine selected from the group consisting of a virus, a bacterium, a mycoplasma, a chlamydia, a viral vector vaccine, a cell vector vaccine, a DNA/mRNA vaccine, a pathogen protein comprising an amino acid sequence and/or a nucleic acid sequence, a candidate protein drug, a candidate DNA or RNA drug molecule, a glycoprotein.
  5. 5. A gene editing system for constructing LgBiT transgenic mice is a CRISPR/Cas system, and comprises an endonuclease Cas, a guide RNA (gRNA) specifically targeting a mouse gene ROSA26 site (NCBI accession number NR_ 027008.1), and a Donor vector containing LgBiT genes, wherein the gene sequence of the gRNA is shown as SEQ ID NO. 5.
  6. 6. The gene editing system according to claim 5, wherein the LgBiT gene is a polynucleotide having a nucleotide sequence shown as SEQ ID NO. 1 or a polynucleotide having a nucleotide sequence having 80% or more homology with SEQ ID NO. 1.
  7. 7. The gene editing system according to claim 6, wherein the Donor vector is pcDNA3.1-LgBiT comprising the following fragments of SEQ ID NO. 29, SEQ ID NO. 30, 0.5 kb Kozak-LgBiT, SEQ ID NO. 31, 0.6 kb WPRE, SEQ ID NO. 32, 0.3 kb BGH pA, SEQ ID NO. 33, 2.2 kb 3' homology arm SEQ ID NO. 34, connected in sequence.
  8. 8. A kit for constructing LgBiT transgenic mice, comprising the gene editing system of any one of claims 5-7, further comprising a microinjection needle for microinjection of Cas protein, gRNA and Donor vectors into mouse fertilized eggs, and a transplantation tube for transplanting fertilized eggs into the uterus of female mice.
  9. 9. The kit of claim 8, further comprising a PCR system for verifying LgBiT gene knockins, said PCR system comprising the following two pairs of primer sequences (1) and (2): (1) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26), Reverse primer (R1): 5'-AGATGTACTGCCAAGTAGGAAAGTC-3' (SEQ ID NO: 27); (2) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26), Reverse primer (R2): 5'-ATACTCCGAGGCGGATCACAA-3' (SEQ ID NO: 28).
  10. 10. The gene editing system according to any one of claims 5 to 7 or the kit according to any one of claims 8 to 9, which is a method for constructing LgBiT transgenic mice, comprising the steps of: 1) Microinjection of Cas protein, gRNA and Donor vector into fertilized eggs of mice; 2) Implanting the microinjected fertilized eggs into oviducts of the surrogate mice for in vivo propagation, and carrying out PCR and sequencing identification after the birth of the mice to obtain LgBiT gene positive F0 mice; 3) Mating the sexually mature positive F0 generation mice with wild mice to reproduce one generation, and carrying out PCR identification after the mice are born to obtain LgBiT gene positive F1 generation heterozygote mice; 4) Inbred propagation is carried out on the F1 generation heterozygote mice, and PCR identification is carried out after the mice are born, so that LgBiT gene positive F2 generation homozygous mice are obtained.

Description

Construction method and application of LgBiT transgenic mice Technical Field The invention belongs to the technical field of medical animal models, and relates to application of a NanoLuc luciferase double subunit complementary system in constructing an experimental animal model, a construction method of LgBiT transgenic mice and a gene editing system for constructing a LgBiT transgenic mouse model. Background The in vivo imaging technology of living animals can track, visualize, characterize and measure physiological changes of the animals in real time in a living and non-invasive state, and qualitative or quantitative research on biological processes of the living animals is realized through an imaging method. According to the technical principle and application scene, the imaging method is mainly divided into two major categories of functional imaging and structural imaging. Wherein the functional imaging includes visible light imaging (optical imaging), nuclear imaging (radio-nuclear imaging), and the structural imaging includes nuclear magnetic resonance imaging (magnetic resonance imaging, MRI), ultrasound imaging (ultrasonic imaging), and computed tomography imaging (computed tomography, CT). Compared with structural imaging suitable for anatomical imaging, functional imaging is more suitable for molecular, metabolic and physiological research, and can more intuitively know relevant biological processes, specific gene functions, interactions and the like in a living animal body by reflecting the spatial and temporal distribution of cell or gene expression. The functional living animal in vivo imaging can observe and track the expression of target cells, target viruses and genes, detect various molecular times simultaneously, optimize the treatment scheme of medicines and genes, observe the curative effect of the medicines from molecular and cellular levels, evaluate the development process of diseases and infections from the whole animal level, and track the time, environment, development and treatment influence of the same animal. Among them, visible light imaging is favored because of its non-invasiveness, high sensitivity, high spatial-temporal resolution, low cost, and adaptability. Visible light imaging living body imaging technology is mainly based on two revolutionary methods of bioluminescence and fluorescence imaging. Bioluminescence imaging ingeniously uses the luciferase gene to label cells, proteins, viruses, etc. of interest, and when the luciferase encounters its substrate luciferin, a chemical reaction occurs, producing a bright signal. Fluorescent imaging uses fluorescent proteins or special dyes as markers, and the markers emit fluorescence under the irradiation of an external excitation light source. The two light signals penetrating through the tissues are captured by the in-vitro high-precision CCD camera device, and are finally converted into clear images through processing, so that researchers can deeply study and analyze the clear images. In contrast, in-vivo visible light imaging technology tracks dynamic changes in the same animal body by recording the same group of experimental animals at different time points, and the obtained data are more visual and real. In addition, the technology does not relate to radioactive substances, has the characteristics of simplicity in operation and the like, and is widely applied to life science, medical research and the like. Bioluminescence is a phenomenon commonly found in nature and can be observed in insects, bacteria, fungi and certain marine organisms, and this luminescence promotes a variety of physiological activities including communication, camouflage, attraction of prey and knock-back of predators. Non-invasive imaging has been widely explored for biomedical applications since the 1990 s. Bioluminescence systems are an enzymatic reaction of the action of a luciferase and a class of substrates that become luciferin, during which a two-component enzymatic reaction converts chemical energy into light energy in the presence of oxygen, effecting the emission of light. Compared with traditional fluorescence imaging, bioluminescence has the obvious advantages of low toxicity, high sensitivity, no need of external light source and high photon yield. The earliest bioluminescence system used in biomedical science was Pyrophorus plagiophthalamus from beetles, however its large size greatly affected the study of small molecules or small compounds. Subsequently, researchers have discovered and developed smaller luciferases, from deep sea shrimp Oplophorus gracilirostris, the smallest luciferase Nanoluciferase so far, nanoLuc (NLuc), which is only 19 kDa in size, is an important complement to bioluminescence tools. The small size makes the marked cells, proteins and viruses have smaller invasiveness to the sample, is beneficial to keeping the natural state of the original sample, and simultaneously NLuc has extremely high luminous intensity an