Construction method and application of cough behavior animal model

Abstract

The invention relates to the field of animal model construction, in particular to a construction method and application of a cough behavior animal model. According to the application of the NTS brain region Synpr < + > neuron activating substance in preparation of the non-human mammal model, the NTS Synpr < + > positive neuron (Synpr < + >) plays a key role in triggering cough reflex, so that the activating substance capable of activating the excitation of the NTS Synpr < + > positive neuron can be used for preparing the non-human mammal model, and the non-human mammal model can be used for preparing the non-human mammal model. Such as a non-human mammalian model of respiratory disease (cough behavior).

Description

Technical Field

[0001] This invention relates to the field of animal model construction, specifically to a method for constructing and using an animal model of coughing behavior. Background Technology

[0002] Coughing is a common airway defense mechanism that helps clear airway secretions, irritants, foreign particles, and microorganisms. The cough reflex is an effective way to keep the airways open. Up to 40% of adult cases of chronic cough lack an identifiable cause or persist despite optimal treatment, and there remains a lack of widely effective treatments for chronic cough or cough allergy syndrome.

[0003] Utilizing robust animal disease models is a valuable tool for revealing the underlying mechanisms of various diseases and facilitating high-throughput screening of potential therapeutic drugs. Past research on cough primarily used animal models such as cats, dogs, and guinea pigs. In recent years, studies have begun using mice as model animals for cough behavior. For example, existing technologies have used guinea pigs to construct animal models of bronchial allergic cough, and, based on the onset characteristics and patterns of bronchial allergic cough, have designed animal models that induce cough with histamine sensitization and citric acid. There are also animal models that induce cough using capsaicin. In other words, most current methods use chemical reagents to induce cough behavior in animal models. However, there are relatively few reports on the use of conditionally transgenic mice to construct animal models of cough behavior.

[0004] The cough reflex is triggered by cough stimuli from the respiratory tract via cough receptors. These receptors receive airway cough receptors via the vagus nerve and are then transmitted to brain regions in the central nervous system, such as the nucleus tractus solitarius (NTS) and paragenomic nucleus (Pa5), before projecting to the cough reflex center. However, how coughing behavior is mediated by vagal nerve afferentization and the NTS remains largely unknown. Therefore, elucidating the specific neuronal subtypes and neural circuits involved in the cough reflex is crucial, as it will contribute to the construction of conditionally transgenic mouse models of coughing behavior. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing an animal model of coughing behavior and its application.

[0006] Therefore, the present invention provides the following technical solution:

[0007] The embodiments disclosed in this invention involve the Synpr brain region of the NTS. + The use of neuronal activating substances in the preparation of non-human mammalian models. This invention discovered the Synpr neuronal activation substance in the NTS brain region. +Neurons are key neuronal subtypes regulating coughing behavior; therefore, they can be targeted by introducing activating substances that can activate NTS-synaptoporin-positive neurons to construct non-human mammalian models. These include, but are not limited to, the non-human mammalian models of respiratory diseases (coughing behavior) of this invention.

[0008] In some embodiments, the activating substance includes Synpr in the NTS brain region. + Products that specifically regulate the transcription of recombinase genes or their protein expression in neurons. Preferably, the products regulating recombinase gene transcription or protein expression include products that increase the transcriptional level of the recombinase gene, or its protein expression level, or its protein bioactivity. Preferably, the products that specifically regulate recombinase gene transcription or protein expression in the NTS brain region include products that increase the transcriptional level of the recombinase gene, or its protein expression level, or its protein bioactivity. + The product that specifically regulates the transcription of recombinase genes or their protein expression in neurons contains the Synpr promoter. Since NTS contains neurons expressing many proteins, this invention has found that activating neurons expressing Synpr can induce coughing. Inserting the recombinase gene into neurons expressing Synpr can specifically activate these neurons. To ensure specific expression of the recombinase in Synpr-expressing neurons, a Synpr gene promoter is designed upstream of the recombinase-encoding gene in the product that regulates recombinase gene transcription or its protein expression. The recombinase gene can be selected from recombinase-encoding genes that can promote the expression of light-sensitive proteins using optogenetic methods. Preferably, the recombinase includes, but is not limited to, the cre recombinase gene from bacteriophages.

[0009] In a preferred embodiment, the NTS brain region Synpr + Use of neuronal activating substances in the preparation of non-human mammalian models of respiratory diseases. More preferably, use in the preparation of non-human mammalian models of acute or chronic coughing behavior. Animals used to construct non-human mammalian models include, but are not limited to, rodents, felines, canines, or primates.

[0010] The embodiments disclosed in this invention relate to a method for constructing a non-human mammalian model, including the Synpr brain region of the NTS. + The activating substances of neurons are transferred to brain regions and / or neurons of non-human mammals.

[0011] In a preferred embodiment, a gene fragment containing the Synpr gene promoter, recombinase gene, post-transcriptional regulatory elements, and polyadenylate sequence is selectively inserted into the genome of a non-human mammal. The recombinase can specifically target the Synpr gene in the NTS brain region. +High expression in neurons; then, in brain regions and / or neurons of a constructed non-human mammalian model, recombinant vectors expressing, but not limited to, light-sensitive protein, hM3D protein, and bacterial sodium channel NaChBac protein (the recombinant vectors include, but are not limited to, adeno-associated virus) were injected, followed by photoactivation, chemical activation, or induction by bacterial sodium channel NaChBac, which could activate Synpr in the NTS brain region. + Neurons induce coughing behavior in non-human mammalian models.

[0012] In a preferred embodiment, the recombinase gene is selected from the cre recombinase gene derived from bacteriophages.

[0013] In a preferred embodiment, the post-transcriptional regulatory element is selected from the post-transcriptional regulatory element WPRE derived from marmot hepatitis virus.

[0014] In a preferred embodiment, the length of the polyadenylated sequence is 232 adenosine nucleotides.

[0015] In a preferred embodiment, the site of insertion into the genome of a non-human mammal is either the H11 site or the Rosa26 site.

[0016] In a preferred embodiment, the gene fragment contains, from 5' to 3', the Synpr gene promoter, recombinase gene, post-transcriptional regulatory element, and polyadenylate sequence.

[0017] In a preferred embodiment, the fixed-point insertion method employs the CRISPR / Cas method.

[0018] In a preferred embodiment, a method for constructing a non-human mammalian model is provided, comprising the following steps: S1, designing gRNA based on the sequence of the H11 site, and simultaneously constructing a homologous recombination vector containing a gene fragment; wherein the gene fragment contains a Synpr gene promoter, a recombinase gene, a post-transcriptional regulatory element, and a polyadenylate sequence.

[0019] S2. GRNA, Cas protein and homologous recombination vector are mixed and the resulting mixture is injected into non-human mammalian zygotes.

[0020] S3. The fertilized eggs after the mixture was injected in step S2 were transferred into the uterus of a pseudopregnant female non-human mammal to produce F0 generation non-human mammal offspring. The offspring were identified and positive F0 generation non-human mammal offspring were obtained.

[0021] S4. Mating the positive F0 generation non-human mammal offspring from step S3 with wild-type non-human mammals for at least one generation to obtain a stable genetic non-human mammal model.

[0022] In some implementations, the animals used to construct non-human mammal models include rodents, felines, canines, or primates.

[0023] In a preferred embodiment, the animal used to construct the non-human mammalian model is selected from at least one of the rodents of the family Muridae; more preferably, the animal is at least one of the genera Rat (Rattus norregicus) and / or at least one of the genera Mouse (Mouse (Mus culus)); even more preferably, the animal is of the genera Mouse (Mouse (Mus culus)).

[0024] In some implementations, the Cas protein includes, but is not limited to, the Cas9 protein.

[0025] In some embodiments, the sequence of the gRNA is as shown in SEQ ID NO.1.

[0026] The embodiments disclosed in this invention relate to a method for constructing a non-human mammalian model of respiratory disease, comprising: inducing the constructed non-human mammalian model using optogenetic methods, chemogenetic methods, or bacterial sodium channels NaChBac.

[0027] In some embodiments, the optogenetic method involves injecting an adeno-associated virus expressing a light-sensitive protein into the brain region and / or neurons of the non-human mammalian model, followed by photoactivation.

[0028] In some embodiments, the chemical genetic method involves injecting an adeno-associated virus expressing the hM3D protein into the brain region and / or neurons of the non-human mammalian model, followed by clozapine CNO induction.

[0029] In some embodiments, in the induction of the bacterial sodium channel NaChBac, an adeno-associated virus expressing the bacterial sodium channel NaChBac protein is injected into the brain region and / or neurons of the non-human mammalian model, followed by induction with capsaicin and / or citric acid.

[0030] In some embodiments, the adeno-associated virus includes a DIO element.

[0031] In some implementations, the adeno-associated virus expressing the light-sensitive protein includes, but is not limited to, AAV-DIO-ChR2-mCherry adeno-associated virus.

[0032] In some implementations, adeno-associated viruses expressing the hM3D protein include, but are not limited to, AAV-DIO-hM3D-mCherry adeno-associated virus.

[0033] In some embodiments, adeno-associated viruses expressing the bacterial sodium channel NaChBac protein include, but are not limited to, AAV-DIO-NaChBac-EGFP adeno-associated virus.

[0034] In a preferred embodiment, the injection dose of AAV-DIO-ChR2-mCherry adeno-associated virus is 200 nl, and the transfection time is 2 weeks.

[0035] In a preferred embodiment, the injection dose of AAV-DIO-hM3D-mCherry adeno-associated virus is 200 nl, and the transfection time is 2 weeks.

[0036] In a preferred embodiment, the injection volume of AAV-DIO-NaChBac-EGFP adeno-associated virus is 200 nl, and the transfection time is 2 weeks.

[0037] In some implementations, the injection method is stereotactic brain injection.

[0038] In some embodiments, the conditions for photoactivation include, but are not limited to, photoactivation using a 473nm light wave (5Hz, 1-5mW, 5ms, 5s).

[0039] In some embodiments, the CNO induction is performed by intraperitoneal injection of CNO at a concentration of 0.01 mg / kg, which is five times the body weight of the mouse.

[0040] In some embodiments, the capsaicin and / or citric acid induces / activates the mice by preparing 0.01 mM capsaicin and / or citric acid and inhaling it through a nebulizer to induce coughing behavior in mice.

[0041] In a preferred embodiment, the non-human mammalian model of respiratory disease is a non-human mammalian model of acute or chronic cough.

[0042] The embodiments disclosed in this invention relate to a non-human mammalian model constructed using the aforementioned method for constructing a non-human mammalian model or the aforementioned method for constructing a non-human mammalian model of respiratory diseases.

[0043] The embodiments disclosed in this invention relate to a method for screening or identifying drugs that can alleviate or treat respiratory diseases using a non-human mammalian model constructed by the aforementioned method for constructing a non-human mammalian model, comprising:

[0044] In the experimental group, the test product was applied to a non-human mammalian model of the respiratory disease; a control group was set up, which was treated with a solvent that did not contain the test product.

[0045] Comparing the experimental group and the control group, the experimental group showed improved respiratory disease characteristics in non-human mammalian models of respiratory diseases treated with the test product, indicating that the test product has the effect of alleviating or treating respiratory diseases.

[0046] The technical solution of this invention has the following advantages:

[0047] 1. The NTS brain region Synpr provided by this invention + The use of neuronal activators in the preparation of non-human mammalian models: This invention has discovered and confirmed that NTS Synaptoporin-positive neurons (Synpr+) play a key role in triggering the cough reflex. Therefore, activators that can activate NTS Synaptoporin-positive neurons can be used to prepare non-human mammalian models, such as non-human mammalian models of respiratory diseases (coughing behavior).

[0048] 2. The present invention provides a method for constructing a non-human mammalian model, comprising: synthesizing the NTS brain region Synpr... + The activating substances of neurons are transferred into brain regions and / or neurons of non-human mammals; this invention studies and confirms that by using CRISPR / Cas9 technology to insert activating substances, such as the Synpr-iCre gene fragment, into the H11 site of mice, a mouse model that can stably induce cough-like behavior was established through mouse neural manipulation technology.

[0049] 3. The present invention provides a method for constructing a non-human mammalian model of respiratory diseases, comprising inducing the non-human mammalian model (transgenic Synpr-iCre gene) using optogenetic methods, chemogenetic methods, or bacterial sodium channels NaChBac; the above methods can induce coughing behavior in mouse models, which on the one hand shows that the non-human mammalian model (transgenic Synpr-iCre gene) has a wide range of applications and can be induced by different methods; on the other hand, it has been verified that the coughing behavior induced by the non-human mammalian model (transgenic Synpr-iCre gene) is almost identical to the cough-like behavior induced by capsaicin in wild-type mice, indicating that the non-human mammalian model (transgenic Synpr-iCre gene) can be used to construct animal models of coughing behavior for the development of mechanisms and drugs for acute or chronic cough and other related respiratory diseases, including screening and identification. Attached Figure Description

[0050] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0051] Figure 1 This is the screening of neuronal subtypes regulating cough behavior in Embodiment 1 of the present invention; a) The left UMAP shows NTS neuronal clusters, including excitatory neurons (orange) and inhibitory neurons (blue); the right figure shows four NTS excitatory neuronal clusters of different colors; b) A heatmap of the first 15 marker genes (y-axis) of each cluster (x-axis) in mouse NTS neurons; c) A schematic diagram of the method for constructing AAV-DEGsProm-Cre virus; d) A schematic diagram of the virus injection and fiber optic implantation sites; e) The total number of light-induced coughs of the first 15 marker genes in mice;

[0052] Figure 2 This is the construction of the Synpr-iCre mouse model in Example 2 of the present invention; a) homologous recombination plasmid map; b) schematic diagram of CRISPR-Cas9 technology for constructing Synpr-iCre model mice; c) Synpr-iCre mouse DNA fragment bands, with bands numbered 14, 16, and 19 representing Cre-positive mice, and no band numbered 20 representing wild-type wt mice.

[0053] Figure 3a This invention relates to the audio spectrum analysis of cough audio from an animal model in Experiment Example 1, which simultaneously records and analyzes the WBP, electromyography, and audio sequence of wild-type mice coughed inducing by capsaicin.

[0054] Figure 3b This invention relates to the audio spectrum analysis of cough audio in an animal model in Experiment Example 1, which includes the synchronous recording and analysis of WBP, electromyography, and audio sequence of photoactivated Synpr-iCre mice during coughing.

[0055] Figure 3c This is a statistical analysis of the average WBP value of the animal model cough audio in Experiment Example 1 of this invention when the cough was induced by capsaicin in mice.

[0056] Figure 3d This is a statistical analysis of the average WBP value of the cough audio of the animal model in Experiment Example 1 of this invention when coughing was induced by light activation of Synpr-iCre mice;

[0057] Figure 4Examples of the invention include: a) the total number of chemically activated NTS Synpr+ neurons; b) examples of action potential discharges demonstrating the effectiveness of CNO in chemically activating NTS neurons expressing hM3D; c) a curve of peak activity induced by depolarization of EGFP+synpr neurons infected with NTS; d) quantitative analysis of c; e) coronal brain slices showing the expression of AAV-DIO-NaChbac-EGFP in the NTS of Synpr-iCre mice; f) the total number of coughs induced by capsaicin or citric acid in Synpr-iCre mice expressing NaChBac or EGFP in Synpr+NTS neurons. Detailed Implementation

[0058] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0059] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0060] As used in this article, Synpr+ can be replaced with Synaptoporin.

[0061] Optogenetics induces coughing behavior. This technique utilizes gene manipulation to introduce exogenous light-sensitive protein genes into living cells, expressing light-sensitive channel proteins on the cell membrane structure. Then, irradiation with specific wavelengths of light activates and deactivates these light-sensitive channel proteins, controlling the opening and closing of ion channels and altering cell membrane voltage, such as depolarization and hyperpolarization. When membrane voltage depolarization exceeds a certain threshold, it induces the generation of transmissible electrical signals in neurons, i.e., neuronal activation. This study induced coughing behavior by expressing light-sensitive channel proteins in Synpr+ neurons in the NTS brain region and activating these neurons through irradiation with specific wavelengths of light to control the opening and closing of ion channels.

[0062] Chemogenetic methods were used to induce coughing behavior. The DREADDs technology alters the structure of the G protein-coupled receptor—acetylcholine receptor—so that it can only be activated or inhibited by a specific compound, Clozapine-N-oxide (CNO). In this study, by expressing hM3Dq in Synpr+ neurons in the NTS brain region, and then activating these neurons with peritoneal CNO three weeks later, coughing behavior was induced in mice.

[0063] NaChBac is the first Na+ channel characterized as a voltage-gated channel, capable of selectively gating Na+ in response to changes in the cell membrane potential. + It penetrates the entire cell membrane to initiate and propagate action potentials. Expression of NaChBac in neurons can generate long-duration action potentials and depolarizations lasting hundreds of milliseconds. Currently, expressing bacterial sodium channels (NaChBac) in target neurons to achieve chronic activation of neuronal activity has been widely used in neuroscience research. In this study, hM3Dq was expressed in Synpr+ neurons in the NTS brain region, and coughing behavior was induced in mice by inhaling capsaicin or citric acid via nebulization.

[0064] Preparation method of AAV-DEGsProm-Cre virus: The target gene was subcloned into the target AAV expression vector through a simple enzyme digestion-ligation-transformation method. Positive clones were obtained, and sequencing was used to confirm the correctness of the inserted fragment. Purchased from Priscilla Technologies.

[0065] The AAV-DIO-ChR2-mCherry virus expressing the photosensitive protein was purchased from Priscilla Technologies.

[0066] The AAV-DIO-hM3D-mCherry virus was purchased from Privy Technologies.

[0067] The AAV-DIO-NaChBac-EGFP virus was purchased from Primacy Technologies. The sequences involved in the following examples are shown in the table below:

[0068] Table 1, Sequence

[0069]

[0070] Example 1: Screening of neuronal subtypes regulating cough behavior

[0071] (1) Single-cell RNA sequencing (scRNA-seq) was performed on neurons in the NTS brain region of mice (conducted by Annoroad Gene Technology (Beijing) Co., Ltd.). scRNA-seq mainly includes four steps: 1) isolation or lysis of single cells or single cell nuclei; 2) reverse transcription; 3) cDNA amplification; and 4) library construction and sequencing. The sequencing results identified 15 highly expressed differentially expressed gene (DEG) types (e.g., Figure 1 (As shown in a and b).

[0072] (2) Screening of key neuronal subtypes for cough behavior. Fifteen differentially expressed genes (DEGs) were selected from the sequencing results and inserted into AAV adeno-associated virus to synthesize AAV-DEGsProm-Cre virus (e.g., Figure 1 c) Fifteen AAV-DEGsProm-Cre viruses were mixed with AAV-DIO-ChR2-mCherry viruses expressing light-sensitive proteins (each in a concentration of 2 x 10⁻⁶). 12 PFU (200 nmol) was injected into the NTS brain region of wild-type mice using a stereotaxic device (e.g., Figure 1 (d) Synpr was selected through photo-activated screening. + Neurons may be key neurons in inducing the cough reflex (e.g.) Figure 1 In the middle (e), you can see Synpr + A high bar indicates a high frequency of coughing behavior induced in mice, suggesting that NTS Synaptoporin-positive neurons (Synpr) are more likely to induce coughing. + It may play a key role in triggering the cough reflex.

[0073] Example 2: Construction of Synpr-iCre transgenic mouse model

[0074] H11, located on mouse chromosome 11, is a safe site for the insertion of foreign genes. Foreign genes integrated into this site can be expressed stably and efficiently without disrupting the function of endogenous genes. Therefore, in this embodiment, CRISPR-Cas9 technology was used to insert the Synpr-iCre-Wpre-PolyA gene fragment (5'→3' sequentially Synpr promoter, iCre, WPRE, PolyA, as shown in SEQ ID NO. 1, 1641bp~5526bp) into the mouse H11 site (e.g., ...). Figure 2 In section b, the H11 gene sequence after insertion of the Synpr-iCre-Wpre-PolyA gene fragment is shown in SEQ ID NO.1. The homologous recombination plasmid map is shown below. Figure 2 As shown in Figure a, the specific steps include the following:

[0075] (1) Construction of gRNA and homologous recombination vector

[0076] gRNA was designed, constructed, and transcribed in vitro based on the H11 gene sequence, see SEQ ID NO.2.

[0077] Homologous recombination vector (Donor vector) (e.g.) Figure 2 a) contains the Synpr-iCre-Wpre-PolyA gene fragment, which can be prepared using conventional methods in the field. In this embodiment, it was synthesized by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0078] (2) Microinjection

[0079] CRISPR-Cas9 and the homologous recombinant vector were mixed and the concentrations were adjusted to obtain a mixture with Cas9 protein concentration of 400 ng / ul, gRNA concentration of 200 ng / ul, and homologous recombinant vector concentration of 20 ng / ul. The mixture was then microinjected into C57BL / 6JGpt mouse zygotes using a microinjector with an injection volume of 20 pL. The zygotes were then transferred into the uterus of pseudopregnant female mice, and the mice were left to grow into the F0 generation.

[0080] (4) Identification of F0 generation mice.

[0081] The F0 generation pups born to the recipient mice were tail-cropped and toe-cropped at 5-7 days old for numbering. Genomic DNA was extracted for PCR and sequencing identification (see step (6)). The genotype was confirmed (containing a 1381bp band, indicating cre positive), and positive F0 generation mice were obtained.

[0082] (5) Breeding of positive F0 generation mice

[0083] After reaching sexual maturity, positive F0 generation mice were mated with wild-type background mice (C57BL / 6JGpt mice, 10 weeks old, weighing 23-25g). The resulting F1 generation mice were tail-cropped and toe-cropped at 5-7 days of age for numbering. Genomic DNA was extracted for PCR and sequencing to confirm genotype and obtain stably heritable F1 generation positive mice (e.g., F0 mice). Figure 2 c) mouse model, photographic results as follows ( Figure 2 As shown in c), samples 14, 16, and 19 containing a 1381bp band were cre positive, and sample 20 with five bands was wild-type (wt). The F1 generation of cre positive mice that can be stably inherited was named Synpr-iCre mouse.

[0084] (6) Identification of Synpr-iCre mice

[0085] 1) Cells are lysed and proteins are digested using proteinase K and SDS in the presence of EDTA. This process depolymerizes nucleoproteins and inactivates intracellular DNases. After extraction with phenol-chloroform organic solvent, purified DNA is obtained by precipitation with ethanol. This ensures the integrity of the primary structure of nucleic acids and eliminates contamination from other molecules, minimizing protein / sugar / RNA contamination.

[0086] 2) Digestive system

[0087] Prepare the mouse tissue digestion solution according to Table 2, add it to a 1.5 ml EP tube containing mouse tail tissue, and digest at 55°C for 12 h.

[0088] Table 2. Tissue digestion fluids

[0089]

[0090] 3) Purification operation steps

[0091] Take 200 μL of the lysed product (if digestion is incomplete and tissue or hair residue remains, briefly centrifuge and collect the supernatant first), add 2 volumes (400 μL) of anhydrous ethanol, and gently invert 10-15 times to mix. White flocculent DNA should be visible at this point. Centrifuge at 12000 rpm for 5 minutes. Discard the supernatant after centrifugation, add 400 μL of 70% ethanol, wash by inverting 10-15 times, and centrifuge at 12000 rpm for 5 minutes. Discard the supernatant after centrifugation, and briefly centrifuge the centrifuge tube at 12000 rpm for 30 seconds. Carefully remove the residual ethanol with a pipette, open the centrifuge tube, and let it air dry at room temperature for 20-30 minutes. Add 100-200 μL of deionized water to dissolve the DNA (adjust the amount of dissolving solution according to the amount of template, but at least 30 μL).

[0092] 4) Construction of PCR system

[0093] Prepare the PCR reaction system according to the three cells in Table 3 below, and add the DNA template extracted in the above steps.

[0094] Table 3. PCR System

[0095] step Reaction element Volume (ul) 1 2×Mix 12.5 2 <![CDATA[ddH2O]]> 9.5 3 Primer A (10 pmol / μl) 1 4 Primer B (10 pmol / μl) 1 5 Nucleic acid model with an initial concentration of 10–100 ng / μl 1

[0096] 5) PCR amplification

[0097] Place the prepared PCR reaction system into a PCR instrument and set the PCR amplification steps as shown in Table 4 below. Primers for the identification of Synpr-iCre mice are shown in Table 1, primers A and B.

[0098] Table 4. Amplification Procedure

[0099]

[0100] 6) Identification of PCR products by agarose gel electrophoresis

[0101] Gel preparation: Weigh 2g of agarose powder using an electronic analytical balance and pour it into an Erlenmeyer flask. Add 100mL of 0.5xTBE electrophoresis buffer and heat in a microwave oven to melt the agarose, preparing a 2% agarose gel. Cool the melted agarose to 60-70℃, add 2µL of nucleic acid dye, and gently shake to mix. Pour the mixture into a gel bath and let it stand at room temperature for about 30 minutes until the gel solidifies.

[0102] Electrophoresis: After the gel solidifies, pull the comb out vertically and place the gel plate into the electrophoresis tank; take 10uL of DNA PCR product and marker, and add them to the gel wells in sequence. According to the distance between the positive and negative electrodes of the electrophoresis tank, calculate the voltage to be used at 3-5V / cm, which is 110V. Electrophoresis for 35min, and then observe and take pictures of the results in the BIO RAD Universal Hood II gel imaging system.

[0103] Example 3: Construction of a Synpr-iCre mouse model of photoactivated cough-like behavior

[0104] Stereotactic injection: Synpr-iCre mice constructed in Example 2 were anesthetized via intraperitoneal injection with tribromoethanol (125-250 mg / kg) solution. After anesthesia, the mice's head hair was shaved to expose the skin, and the mice were fixed to a stereotactic instrument. The scalp was cut open to expose the skull. After leveling with the stereotactic instrument, 200 nmol of AAV-DIO-ChR2-mCherry virus (titer 2 x 10⁻⁶) was injected. 12 PFU was injected into the NTS brain region, and then an optical fiber (core diameter: 200 μm) was inserted above the NTS brain region using a stereotaxic instrument and fixed to the mouse skull with dental cement. A mouse model of coughing behavior was obtained two weeks after infection.

[0105] Coughing behavior in a mouse model induced by photoactivation was induced. The photoactivation conditions were: activation with an optogenetic light source (Reward), and recording of the mouse coughing behavior. The photoactivation conditions were: 473nm light wave with a frequency of 5Hz, a wavelength of 5ms, an energy of 1-5mW, and a stimulation duration of 5s.

[0106] Comparative Example 1: Wild-type mouse model of capsaicin-induced cough-like behavior

[0107] Mice were anesthetized with tribromoethanol (125-250 mg / kg). C57BL / 6J mice, aged 10-15 weeks and weighing 22-25 g, were selected from wild-type mice using a capsaicin-induced cough-like behavior model. The abdomen was then incised to expose the diaphragm. The insulating layer of two spiral tungsten microelectrodes (Am Systems, 795500) was removed from their tips (0.2 mm), and the microelectrodes were inserted into the muscles of the left diaphragm and external oblique muscle using a syringe needle as a guide. The microelectrodes were then extended from the mouse subcutaneously to the head, and a connection was established at the top of the skull. Mice were placed in a WBP chamber, and a whole-body volumetric plethysmography (WBP) device, an electromyography (EMG) recording device (using an 1800 microelectrode AC amplifier (AM Systems) to record EMG signals, filtered (100-5000Hz EMG signals), and Spike2 software (Cambridge Electronic Design) at a 5000Hz sampling rate) were connected, along with an ultrasound recording device (an ultrasound microphone (Avisoft-Bioacoustics CM16 / CMPA) inserted through a hole in the top of the chamber cover to record sound), and a high-speed video recording device. Coughing behavior in mice was induced by continuous nebulization of 0.01mM capsaicin using a nebulizer (Yuwell, M105).

[0108] Experiment Example 1: Verification of Cough-like Behavior

[0109] The Synpr-iCre mice in Example 3 were photoactivated to induce cough-like behavior. The cough-like behavior was recorded in the same way as the capsaicin-induced cough-like behavior of wild-type mice in Comparative Example 1: The mice were placed in a WBP chamber and connected to a whole-body volumetric plethysmography (WBP) device, an electromyography (EMG) recording device (using an 1800 microelectrode AC amplifier (AM Systems) to record EMG signals, filtering (100-5000Hz EMG signals), and using Spike2 software (Cambridge Electronic Design) at a 5000Hz sampling rate), an ultrasound recording device (an ultrasound microphone (Avisoft-Bioacoustics CM16 / CMPA) was inserted through a hole in the top of the chamber cover to record sound), and a high-speed video recording device.

[0110] Comparison of capsaicin-induced cough-like behavior records from wild-type mice (n=4) and photoactivation-induced cough-like behavior records from Synpr-iCre mice (n=4) revealed that the photoactivation-induced cough-like behavior in Synpr-iCre mice and the capsaicin-induced cough-like behavior in wild-type mice not only shared similar WBP respiratory curves (e.g., Figures 3c-3d (Statistical results of 4 mouse models in each group) showed that they were almost identical in terms of electromyography and sound frequency (e.g. Figures 3a-3bThe figures represent the behavior of one mouse model, and other mice in each group showed similar results. This indicates that photoactivation of Synpr-iCre mice reliably induced cough-like behavior.

[0111] Example 4: Construction of the Synpr-iCre mouse model of cough behavior induced by chemogenetic methods and sodium ion channels

[0112] Synpr-iCre mouse model of coughing behavior induced by chemogenetic methods:

[0113] Stereotactic injection: Synpr-iCre mice constructed in Example 2 were anesthetized via intraperitoneal injection with tribromoethanol (125-250 mg / kg) solution. After anesthesia, the mice's head hair was shaved to expose the skin, and the mice were fixed to a stereotactic instrument. The scalp was cut open to expose the skull. After leveling with the stereotactic instrument, 200 nmol of AAV-DIO-hM3D-mCherry (2 x 10⁻⁶ titers) was injected. 12 PFU was injected into the NTS brain region. Two weeks later, CNO was used for activation (intraperitoneal injection of CNO (0.01 mg / kg)), and coughing behavior in mice was recorded.

[0114] Synpr-iCre mouse model of sodium ion channel-induced coughing behavior:

[0115] Stereotactic injection: Synpr-iCre mice constructed in Example 2 were anesthetized via intraperitoneal injection with tribromoethanol (125-250 mg / kg) solution. After anesthesia, the mice's head hair was shaved to expose the skin, and the mice were fixed to a stereotaxic instrument. The scalp was cut open to expose the skull. After leveling with the stereotaxic instrument, 200 nmol of AAV-DIO-NaChBac-EGFP (titer 2 x 10⁻⁶) was injected. 12 PFU was injected into the NTS brain region. Two weeks later, coughing behavior was induced by nebulization of 0.01 mM capsaicin, and the coughing behavior of mice was recorded.

[0116] The cough sensitivity of five Synpr-iCre mouse models (n=12) induced by chemogenetic methods and induced by sodium ion channels was tested. The test method was as follows: the mice were placed in a WBP chamber and connected to a whole-body phrasing (WBP) device, an electromyography (EMG) recording device (using an A-MSystems 1800 microelectrode AC amplifier to record EMG signals, filtered (100-5000Hz EMG signals), and Spike2 software (Cambridge Electronic Design) at a 5000Hz sampling rate), an ultrasound recording device (an ultrasound microphone (Avisoft-Bioacoustics CM16 / CMPA) inserted through a hole in the top of the chamber cover to record sound), and a high-speed video recording device.

[0117] The results are as follows Figure 4 As shown, in the Synpr-iCre mouse model (n=4) induced by chemogenetic methods, coughing behavior could be induced in the mice (e.g., Figure 4 In the figure, *** indicates p < 0.001, and the figures show the statistical results of 4 mice in each group (Ctrl represents the control group, wild-type mice). Simultaneously, in vitro neurophysiological experiments also showed that neurons expressing hM3D in the NTS exhibited significantly increased activity after CNO induction (4 mice in each group, e.g., ...). Figure 4 (Figure b shows representative results from one mouse model; other mice showed similar results). In the sodium ion channel-induced coughing behavior Synpr-iCre mouse model (NaChBac group), AAV-DIO-NaChBac-EGFP virus or AAV-DIO-EGFP was injected into the Synpr+NTS brain region of Synpr-iCre mice (e.g., ...). Figure 4 Figure e shows the representative results of a Synpr-iCre mouse model injected with AAV-DIO-NaChBac-EGFP virus; other mice showed similar results. It was found that the number of coughs induced by capsaicin and citric acid (constructed according to the aforementioned Synpr-iCre mouse model of sodium ion channel-induced coughing behavior, the difference being the use of citric acid) in the NaChBac group was significantly higher than that in the control group (Synpr-iCre mice injected with AAV-DIO-EGFP). Figure 4 In the figure, **** indicates p < 0.0001, and * indicates p < 0.05. The figure shows the statistical results of 6 mice in each group. Simultaneously, in vitro neurophysiological experiments also showed that the frequency of nerve current discharge in the NaChBac group was significantly higher than that in the control group (e.g., ...). Figure 4 c and d, Figure 4Figure d shows the statistical results of 4 mice in each group, and Figure c shows the representative behavior of 1 mouse in each group. Other mice also showed similar results. CON is the control group (Synpr-iCre mice injected with AAV-DIO-EGFP).

[0118] The above demonstrates that the Synpr-iCre mouse model of cough behavior induced by chemogenetic methods and sodium ion channels was successfully constructed.

[0119] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. NTS brain region Synpr + The use of neuronal activating substances in the preparation of non-human mammalian models.

2. The use according to claim 1, characterized in that, The activating substance is included in the Synpr brain region of the NTS. + Products that specifically regulate the transcription of recombinase genes or the expression of their proteins in neurons.

3. The use according to claim 2, characterized in that, The products that regulate recombinase gene transcription or protein expression include products that increase the transcription level of recombinase genes, or their protein expression level, or their protein bioactivity. Optionally, the Synpr in the NTS brain region + In products that specifically regulate the transcription of recombinase genes or their protein expression in neurons, a Synpr gene promoter is designed upstream of the recombinase encoding gene; Optionally, the recombinase gene is selected from the cre recombinase gene from bacteriophages.

4. The use according to claim 2, characterized in that, The uses include those for preparing non-human mammalian models of respiratory diseases; And / or, the non-human mammalian model includes rodents, felines, canines, or primates.

5. The use according to claim 2, characterized in that, The uses include those for preparing non-human mammalian models of acute or chronic cough behavior.

6. A method for constructing a non-human mammalian model, characterized in that, Includes the NTS brain region Synpr as described in any one of claims 1-5 + The activating substances of neurons are transferred to brain regions and / or neurons in non-human mammals.

7. The method for constructing a non-human mammalian model according to claim 6, characterized in that, The non-human mammalian models include rodents, felines, canines, or primates.

8. A method for constructing a non-human mammalian model of a respiratory disease, characterized in that, include: The non-human mammalian model described in any one of claims 6-7 is induced by optogenetic, chemogenetic, or bacterial sodium channel NaChBac methods.

9. The method for constructing a non-human mammalian model of respiratory diseases according to claim 8, characterized in that, In the optogenetic method described above, an adeno-associated virus expressing a light-sensitive protein is injected into the brain region and / or neurons of the non-human mammalian model as described in any one of claims 6-7, followed by photoactivation. Alternatively, in the chemical genetic method, an adeno-associated virus expressing the hM3D protein is injected into the brain region and / or neurons of a non-human mammalian model according to any one of claims 6-7, followed by clozapine induction; Alternatively, in the induction of the bacterial sodium channel NaChBac, an adeno-associated virus expressing the bacterial sodium channel NaChBac protein is injected into the brain region and / or neurons of a non-human mammalian model as described in any one of claims 6-7, followed by induction with capsaicin and / or citric acid.

10. The method for constructing a non-human mammalian model of respiratory disease according to any one of claims 8-9, characterized in that, The respiratory illness mentioned is an acute or chronic cough.

11. A non-human mammalian model constructed by the method for constructing a non-human mammalian model according to any one of claims 6-7 or the method for constructing a non-human mammalian model of respiratory diseases according to any one of claims 8-10.

12. A method for screening or identifying drugs that can alleviate or treat respiratory diseases using a non-human mammalian model constructed according to any one of claims 8-10, characterized in that... include: In the experimental group, the test product was applied to a non-human mammalian model of the respiratory disease; a control group was set up, which was treated with a solvent that did not contain the test product. Comparing the experimental group and the control group, the experimental group showed improved respiratory disease characteristics in non-human mammalian models of respiratory diseases treated with the test product, indicating that the test product has the effect of alleviating or treating respiratory diseases.

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