Construction and application of Primrose syndrome non-human animal model

Abstract

The invention discloses construction and application of a Primrose syndrome non-human animal model, and belongs to the technical field of animal models and disease research. According to the invention, through a CRISPR / Cas9 technology, a codon CAC for coding 596th histidine in a No.14 exon of a mouse Zbtb20 gene is subjected to site-directed mutagenesis into CGC for coding arginine, and a hybrid mouse model for simulating pathogenic point mutation (p.H596R) of human Primrose syndrome is constructed. The model can stably reproduce key clinical phenotypes of the Primrose syndrome, including overgrowth after adult, serum IGF-1 rise, memory dysfunction and anxiety behaviors, and shows abnormal hippocampal neuronal development and synaptic transfer related pathways and the like. The invention provides an important experimental tool for deeply revealing the pathological mechanism of Primrose syndrome, developing drug screening and treatment intervention research and the like.

Description

Technical Field

[0001] This invention belongs to the field of animal model and disease research technology, specifically relating to the construction and application of a non-human animal model of Primrose syndrome. Background Technology

[0002] Primrose syndrome (PS) is a rare genetic disorder, with over 50 cases reported since its first report in 1982. The syndrome presents with diverse clinical manifestations, primarily involving the neuropsychiatric system, growth and development, and metabolism. Neuropsychiatric abnormalities are its most prominent feature; all patients exhibit varying degrees of intellectual disability, and some also present with autism, anxiety, depression, or schizophrenia. Growth and developmental abnormalities vary; most patients are tall or show excessive growth, while a few are short in stature. Metabolic abnormalities are common, including impaired blood glucose homeostasis, potentially leading to diabetes or impaired glucose tolerance in adulthood. Other common features include hearing loss, auricular calcification, cataracts, corpus callosum agenesis, and hypothyroidism.

[0003] ZBTB20 The gene encodes a transcription factor containing a BTB / POZ domain and a C2H2-type zinc finger structure. De novo heterozygous point mutations (non-hereditary) in this gene are the pathogenic basis of Primrose syndrome. Mutations are mainly concentrated in the C-terminal first to third zinc finger domains and their connecting regions. Currently reported pathogenic mutations include 37 point mutations and 5 small deletions or frameshift mutations. Multiple mutation forms exist at some amino acid sites, such as mutations in conserved cysteine ​​or histidine residues within the zinc finger domains, which are speculated to disrupt the structure and function of the ZBTB20 protein. Figure 1 ).

[0004] Patients with Primrose syndrome express one normal copy of the ZBTB20 protein, but differ from patients with 3q 13.31 microdeletion syndrome who also express only one normal copy of the ZBTB20 protein (existing...). ZBTB20 Compared to individuals with loss of heterozygosity, patients with Primrose syndrome exhibit more severe intellectual disability. Similarly, Zbtb20 The behavioral phenotypes of heterozygous knockout mice were very mild. These results indicate that haplo-insufficiency of ZBTB20 alone is insufficient to cause disease, suggesting that the mutant ZBTB20 protein in Primrose syndrome patients may exert some negative regulatory effect on the normal ZBTB20 protein, preventing it from performing its function. Therefore, using... Zbtb20Gene knockout heterozygous mice cannot reflect the biochemical mechanisms of Primrose syndrome, nor can they truly mimic its pathological characteristics, and they are unsuitable for studying the dominant-negative effect of the ZBTB20 mutant protein on normal proteins. Due to the lack of animal models that can adequately mimic the disease characteristics of Primrose syndrome, our understanding of the pathophysiological mechanisms of Primrose syndrome is currently very limited. Summary of the Invention

[0005] Based on the shortcomings of the existing technologies, this invention has for the first time successfully established a heterozygous mouse model with Zbtb20 point mutation, and systematically analyzed its phenotypic characteristics and related mechanisms, providing experimental evidence for revealing the pathological characteristics and pathogenesis of Primrose syndrome, and providing a reliable animal model for the pathogenesis and intervention research of this syndrome.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a non-human animal model of Primrose syndrome. Zbtb20 The gene contains a c.1787 A>G point mutation, which leads to a p.H596R amino acid substitution in its encoded protein.

[0007] Furthermore, the animals include mice.

[0008] The second objective of this invention is to provide a method for constructing a non-human animal model of Primrose syndrome, comprising the following steps: S1. Design and prepare targeted Zbtb2 sgRNA in the intron regions flanking exon 14 of gene 0; S2. Construct a homologous recombination targeting vector containing the c.1787 A>G point mutation site; S3. Inject the Cas9 mRNA, the sgRNA prepared in step S1, and the targeting vector constructed in step S2 into the fertilized egg. S4. The injected fertilized eggs were transferred into pseudopregnant mice to obtain F0 generation mice. S5. Genotyping of F0 generation mice was performed to screen out individuals carrying the c.1787 A>G point mutation.

[0009] Furthermore, in step S1, the sgRNA is screened for activity using the luciferase reporter gene method, and sgRNAs with high cleavage activity are selected for subsequent experiments.

[0010] Furthermore, in step S2, the homologous recombination targeting vector includes... Zbtb20The left and right homologous arms flanking the gene target site, and the sequence containing the c.1787 A>G point mutation located between the homologous arms.

[0011] Furthermore, in step S5, the genotype identification includes: extracting mouse genomic DNA, amplifying the Zbtb20 gene fragment containing the c.1787 site by PCR, sequencing the PCR product, and determining the genotype by analyzing the base type before the CATGTTCGT sequence in the sequencing results.

[0012] The third objective of this invention is to provide the application of the aforementioned non-human animal model of Primrose syndrome in the study of the pathophysiological mechanisms of Primrose syndrome.

[0013] The fourth objective of this invention is to provide the application of the aforementioned non-human animal model of Primrose syndrome in the development of products for diagnosing Primrose syndrome.

[0014] The fifth objective of this invention is to provide the application of the aforementioned non-human animal model of Primrose syndrome in screening drugs for the treatment of Primrose syndrome.

[0015] The sixth objective of this invention is to provide the application of the aforementioned non-human animal model of Primrose syndrome in the development of new drugs for the treatment of Primrose syndrome.

[0016] Compared with the prior art, the present invention has the following beneficial effects: Zinc finger proteins were first identified in 2014. Zbtb20 The genetic basis for this syndrome lies in the neonatal heterozygous point mutation of the gene. This invention has long been dedicated to studying the biological function of ZBTB20, providing important experimental evidence for identifying the pathogenic gene of Primrose syndrome, and simultaneously discovering… Zbtb20 Heterozygous mice with gene knockout did not exhibit phenotypic features such as cognitive impairment similar to those seen in Primrose syndrome patients, suggesting that the ZBTB20 point mutant protein plays a crucial role in the development and progression of this syndrome. However, due to the lack of corresponding point mutant animal models, the pathophysiological mechanisms of Primrose syndrome remain poorly understood to date. Therefore, we established... Zbtb20 A heterozygous mouse model of point mutation was developed, and its phenotypic characteristics and related mechanisms were systematically analyzed. It was found that the H596R point mutation heterozygous mouse has phenotypic characteristics such as memory dysfunction, anxiety-like behavior, and excessive growth in adulthood, which are consistent with the clinical manifestations of Primrose syndrome. This provides experimental evidence for revealing the pathological characteristics and pathogenesis of Primrose syndrome, and provides a reliable animal model for the pathogenesis and intervention research of this syndrome. Attached Figure Description

[0017] Figure 1 A summary of ZBTB20 protein point mutations in patients with Primrose syndrome.

[0018] Figure 2 Zbtb20 in this invention H596R A schematic diagram of the point mutation mouse construction strategy (A) and a simplified diagram of the ligation of PCR products amplified by knock-in gene primers (B): The target mutation site is located in exon 14 of the Zbtb20 gene, with the proposed mutation sites being c.1787A>G and p.H596R. sgRNAs were designed in the intron regions flanking exon 14 and cleaved using CRISPR / Cas9 technology. Simultaneously, targeting vectors with homologous arms on both sides and the mutation site in the middle of the cleavage were constructed and inserted into the genome via homologous recombination.

[0019] Figure 3 The results of sgRNA cleavage activity detection in this invention were obtained using UCA. TM The method detects sgRNA cleavage activity by analyzing fluorescence intensity, with sgRNA1 at the 5' end and sgRNA12 at the 3' end showing the strongest activity.

[0020] Figure 4 The following is an agarose gel electrophoresis image of the in vitro transcription of sgRNA1 and sgRNA12 in this invention: before denaturation (without 65°C treatment), sgRNA appears as two bands, while after denaturation treatment (65°C), it appears as one band.

[0021] Figure 5 This is an agarose gel electrophoresis image of the knock-in gene vector after enzyme digestion in this invention: lanes 1-6 represent the numbers of the small plasmids extracted for constructing the targeting vector. After double digestion with SmaI and XhoI, lanes 1-6 show two bands of 6362bp and 1527bp respectively; after digestion with EcoRI, three bands of 4527bp, 2366bp, and 996bp respectively; and after single digestion with HindIII, four bands of 5073bp, 1383bp, 947bp, and 486bp respectively. M: DNA Marker.

[0022] Figure 6 This is a schematic diagram of the point mutation mouse reproduction strategy in this invention.

[0023] Figure 7The results of mouse genotyping in this invention are as follows: (A) Agarose gel electrophoresis of PCR amplification products of mouse tail DNA. (B) Sequencing results of PCR amplification products: wild-type (WT) mice show a green single peak, homozygous (Mut / Mut) mice show a black single peak, and heterozygous (Mut / +) mice show nested double peaks. M: DNA marker.

[0024] Figure 8 Demonstrating the low birth weight and hypoglycemia of Mut / Mut mice in this invention: 3-week-old wild-type mice and ZBTB20 H596R Body weight (A) of point mutation heterozygous Mut / + and homozygous Mut / Mut mice; and random blood glucose (B). p <0.001.

[0025] Figure 9 The present invention demonstrates the abnormal hippocampal cell structure in Mut / Mut mice: (A) Immunohistochemistry of the hippocampus ZBTB20 in 3-week-old wild-type mice and Mut / Mut mice under normal dietary conditions; and (B) Hippocampal Niemann staining showed abnormal hippocampal cell architecture in Mut / Mut mice, with the stratum radiatum (SR) and stratum oriens (SO) on both sides of the pyramidal layer not being identifiable, and the morphology of the DG region of the dentate gyrus missing.

[0026] Figure 10 ZBTB20 is shown in this invention. H596R Point mutation homozygous mouse anterior pituitary PRL + Cell population loss: 3-week-old wild-type WT mice and ZBTB20 were used. H596R Immunohistochemistry of pituitary PRL in homozygous mutant Mut / Mut mice.

[0027] Figure 11 This invention demonstrates the accelerated growth of Mut / + mice in adulthood: (A, A') Mut / + mice and their corresponding littermate wild-type control (WT) mice were collected under normal diets and their body weight and length were measured at 12 weeks of age, (B, B') at 19 weeks of age, (C, C') at 29 weeks of age, and (D, D') at 38 weeks of age. (F) Gross appearance of 29-week-old male mice. Data are presented as mean ± standard error. p <0.05, p <0.01, p <0.001.

[0028] Figure 12 The following diagram illustrates the increased body length and long bone elongation in Mut / + mice as described in this invention: (A) Gross skeletal view of 32-week-old Mut / + mice and wild-type mice. (B) Body length and tibia length of 32-week-old mice. (C) Weight growth curve. p <0.05, p <0.01, p <0.001.

[0029] Figure 13 The invention demonstrates the upregulation of IGF1 protein levels in the serum and liver of Mut / + mice: plasma IGF1 levels were detected by ELISA (A); liver IGF1 mRNA levels were detected by quantitative RT-PCR (B); liver protein levels were detected by Western blotting (C) and their grayscale scanning statistical results (D). p <0.05.

[0030] Figure 14 The invention demonstrates hypothyroidism in Mut / + mice: plasma from 7-month-old mice was collected, and the levels of T4 (A), T3 (B), rT3 (C), TSH (D), and TRH (E) were detected by ELISA. p <0.05.

[0031] Figure 15 The invention demonstrates that no abnormalities were observed in the development of the anterior pituitary cell subsets in Mut / + mice: (A) Seven-month-old male mice were used, and after pituitary fixation, paraffin sections were immunohistochemically analyzed for growth hormone (GH), (B) prolactin (PRL), (C) thyroid-stimulating hormone (TSH), and (D) adrenocorticotropic hormone (ACTH).

[0032] Figure 16 The water maze experiment of this invention demonstrates that Mut / + mice exhibit impaired spatial memory: (A) 3-4 month old male mice were placed in a water maze with a hidden platform (hidden 1 cm below the water surface) and trained for 9 days. The time it took for them to find the platform was observed within 60 seconds. (B, C) On the first day after the training ended, the platform was removed, and the time and proportion of mice crossing the quadrant where the platform was located were observed. (D) Swimming speed, (E) Distance traveled, and (F) Trajectory. p <0.05. n=7.

[0033] Figure 17This invention demonstrates that Mut / + mice exhibit impaired long-term memory (water maze test).

[0034] Figure 18 The following demonstrates the anxiety-like behavior exhibited by Mut / + mice in this invention (open field exercise experiment): (A) Trajectory and heatmap of mice and their wild-type control mice in the open field exercise experiment. The open field was virtually divided into 16 small squares, with the four central squares defined as the central area and the twelve peripheral squares defined as the peripheral area. (B) Distance traveled by mice in the central area and total distance traveled during the open field exercise experiment. (C) Time spent by mice in the peripheral and central areas during the open field exercise experiment. Data are presented as mean ± standard error. , p <0.05, , p <0.01, , p <0.001. n=7.

[0035] Figure 19 The following demonstrates the anxiety-like behavior exhibited by Mut / + mice in this invention (black and white box experiment): (A) Movement trajectories and heat maps of mice and their control wild-type mice in the black and white box experiment. The experimental box consisted of a dark box (left side) and a bright box (right side), connected by a small door. Mice were placed in the bright box and allowed to move freely for 10 minutes. (B) Time spent by mice in the dark and white box areas. (C) Number of times mice entered the bright box. (D) Distance traveled by mice in the dark and white box areas. Data are presented as mean ± standard error. p <0.05. n=7.

[0036] Figure 20 The anxiety-like behavior (elevated cross maze) exhibited by Mut / + mice in this invention is demonstrated: (A) Movement trajectories of mice and wild-type control mice in the elevated cross maze. The experimental cross is divided into open arms (vertical) and closed arms (horizontal). The intersection of the open and closed arms forms a central area. Mice are placed in the central area, facing the open arms, and allowed to move freely. The data is recorded for 10 minutes. (B) Time spent by the experimental mice in the open and closed arms. (C) Number of times the experimental mice opened the arms. Data are presented as mean ± standard error. p <0.05, p <0.01, p <0.001.

[0037] Figure 21 To illustrate the social novelty bias disorder exhibited by Mut / + mice in the second phase of the three-box social experiment in this invention: After the first phase, another unfamiliar mouse (Stranger2) of the same strain, sex, and age was placed in the empty cage of the leftmost box, while the Stranger1 mouse diagonally in the rightmost box remained unchanged. The experimental mice were allowed to continue freely exploring the three boxes. (A) Movement trajectory diagram of Mut / + mice and their control wild-type WT mice in the three-box social experiment. Empty cage (Emp); Stranger1 mouse (S1). (B) Contact time between Mut / + mice and their control wild-type WT mice with Stranger2 and Stranger1 mice in the three-box social experiment. (C) Social novelty bias index. Data are presented as mean ± standard error. p<0.05.

[0038] Figure 22 This invention demonstrates that the ZBTB20 H596R heterozygous point mutation does not affect hippocampal morphology: (A) HE staining of hippocampal sections, where hippocampus 4× (a), a' is an enlarged image of region CA1 in figure a, and hippocampus 10× (b, c). (B) Nissl staining results of hippocampus, where a' is an enlarged image of region CA1 in figure a.

[0039] Figure 23 This demonstrates that the H596R heterozygous mutation in ZBTB20 does not affect the nuclear-cytoplasmic distribution in its hippocampal neurons.

[0040] Figure 24 The results of Golgi staining and Sholl analysis of CA1 region neurons in the mouse hippocampus in this invention are as follows: (A) Brain tissue was taken from 6-month-old Mut / + mice and littermate control mice for Golgi staining. The largest sagittal section of the hippocampus was scanned and photographed to observe the morphology and branches of intact neurons in the field of view. 200x field of view, scale bar = 100 μm. (B) A schematic diagram of the structure of a single hippocampal neuron was drawn using ImageJ software. (C) The number of intersections between the neuron and each concentric circle was counted in the Sholl analysis. n =7 or 18 neurons per group). p <0.05 vs. WT. Mut / + mice: Zbtb20 H596R Heterozygous point mutant mice; WT: wild-type mice from the same littermate.

[0041] Figure 25The results of the dendritic spine density analysis of CA1 region neurons in the mouse hippocampus in this invention are as follows: (A) Brain tissue was taken from 6-month-old Mut / + mice and littermate control mice for Golgi staining. The largest section of the hippocampus was selected for scanning and imaging to observe the density and morphology of the dendritic spines of neurons. 800x field of view, scale bar = 10 μm, red arrows indicate mature mushroom-shaped dendritic spines; (B) Total dendritic spine density statistics ( n =7); (C) Statistical analysis of mature mushroom-like dendritic spine density ( n =7). p <0.05 vs. WT. Mut / + mice: Zbtb20 H596R Heterozygous point mutant mice; WT: wild-type mice from the same littermate.

[0042] Figure 26 The results of the GO enrichment analysis of differentially expressed genes in the Bulk RNA-seq of Mut / + and WT mice in the hippocampus of this invention are shown.

[0043] Figure 27 The results of the KEGG enrichment analysis of differentially expressed genes in the Bulk RNA-seq of Mut / + and WT mice in this invention are shown.

[0044] Figure 28 The results of Hallmark gene set GSEA analysis of hippocampal Bulk RNA-seq in this invention are shown.

[0045] Figure 29 The GSEA analysis in this invention indicated that the oxidative phosphorylation pathway was downregulated in the hippocampus of Mut / + mice.

[0046] Figure 30 This is the t-SNE visualization result of mouse hippocampal scRNA-seq cell clustering in this invention.

[0047] Figure 31 This is the UMAP visualization result of mouse hippocampal scRNA-seq cell clustering in this invention.

[0048] Figure 32 This is a violin diagram of the marker molecules for each cell population in the mouse hippocampus scRNA-seq of this invention.

[0049] Figure 33 This is a dot map of marker molecules in each cell population of the mouse hippocampus using scRNA-seq in this invention.

[0050] Figure 34 The annotation results (tSNE) of each cell population in the mouse hippocampus scRNA-seq in this invention.

[0051] Figure 35This is the annotation result (UMAP) of each cell population in the mouse hippocampus scRNA-seq in this invention.

[0052] Figure 36 This is a profile of the changes in the proportions of different cell populations in the mouse hippocampus using scRNA-seq in this invention.

[0053] Figure 37 The results of the GSEA pathway analysis of the mouse hippocampal scRNA-seq CA1 pyramidal neuron cell population are presented in this invention.

[0054] Figure 38 The invention demonstrates that glutamatergic and synaptic-related pathways are significantly downregulated in the CA1 pyramidal neuronal cell population of the hippocampus in Mut / + mice: (A) positive regulatory pathway of synaptic glutamatergic transmission; (B) synaptic maturation; (C) regulatory pathway of neurotransmitter receptor activity; (D) glutamate receptor signaling pathway.

[0055] Figure 39 The results of the GSEA pathway analysis of the mouse hippocampal scRNA-seq CA3 pyramidal neuron cell population are presented in this invention.

[0056] Figure 40 The invention demonstrates that glutamatergic and synaptic-related pathways are significantly downregulated in the CA3 pyramidal neuronal population of the hippocampus in Mut / + mice: (A) the regulatory pathway of neurotransmitter receptor activity; (B) the regulatory pathway of excitatory postsynaptic potentials; (C) the positive regulation of excitatory postsynaptic potentials; and (D) the regulation of neuronal synaptic plasticity.

[0057] Figure 41 The results of GSEA pathway analysis of mouse hippocampal scRNA-seq granule cell neuronal populations are presented in this invention.

[0058] Figure 42 This invention demonstrates that the excitatory postsynaptic potential pathway is significantly downregulated in hippocampal granule cell neurons of Mut / + mice.

[0059] Figure 43 ZBTB20 is shown in this invention. H596R Mutation leads to impaired hematoxylinization modification of ZBTB20: (A) HEK293A cells were transfected with ZBTB20 wild-type and mutant expression plasmids, with TAK-981 treatment added as a negative control, and the ZBTB20 protein and its hematoxylinization modification level were detected. (B) HEK293A cells were transfected with Flag-SUMO and UBC9, and wild-type ZBTB20 and H596R mutant expression plasmids were transfected, respectively. Anti-Flag antibody was used for IP experiments, and Western blot was used to compare the hematoxylinization modification levels of ZBTB20 wild-type and H596R mutant. Detailed Implementation

[0060] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0061] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0062] Example 1 I. Strategy for Constructing ZBTB20 Point Mutant Mice The 596th His codon in the first zinc finger region of ZBTB20, which is involved in maintaining the C2H2 domain, was selected as the mutation site. Clinically, heterozygous patients with this mutation, who are Arg, exhibit typical manifestations of Primrose syndrome. Using CRISPER / Cas9 technology, sgRNA was directed to bind specifically to the target gene in the introns flanking exon 14, which encodes the 596th His codon. Genomic DNA was then cleaved, and exogenous homologous arm donor DNA was used to mutate the codon CAC encoding the 596th His codon in the Zbtb20 genome to the codon CGC encoding Arg. Figure 2 ).

[0063] 1) Screening and preparation of sgRNA The activity of sgRNA was identified using the luciferase reporter gene assay (UCA) from Biocytogen. TM The activity of sgRNA can be determined by fluorescence intensity. If the sgRNA has strong cleavage activity and high efficiency, the detected fluorescence intensity will be strong. Results are as follows: Figure 3 As shown, among the sgRNAs designed at the 5' end, sgRNA1 has the highest activity, while sgRNA12 at the 3' end has the strongest activity. Therefore, sgRNA1 and sgRNA12 were ultimately selected for subsequent experiments.

[0064] sgRNA1 and sgRNA12, which exhibit high cleavage activity, were screened and annealed to form two pairs of upstream and downstream oligonucleotides, respectively. A BsmI restriction site and the prefix TAGG were added to the 5' end of the upstream primer, and CAAA was added to the downstream primer. After synthesizing the oligonucleotide chains, two sgRNA1 and sgRNA12 dimers were synthesized by annealing. The dimerized sgRNA was ligated to a BsmI-digested PT7-sgRNA plasmid, transformed, and subjected to bacterial selection, culture, and plasmid micro-sampling. Sequencing confirmed the correct insertion of the PT7-sgRNA1 (or PT7-sgRNA12) recombinant plasmid. Using the T7 promoter on the pT7 plasmid, the PT7-sgRNA1 (or PT7-sgRNA12) recombinant plasmid was transcribed in vitro and purified to obtain sgRNA, which was then identified by agarose gel electrophoresis. As shown in Figure 4, sgRNA1 and sgRNA12 were successfully transcribed in vitro.

[0065] 2) Construction of the target carrier like Figure 5 As shown, six clones were selected and digested with enzymes. The bands of plasmids 1# to 6# were correct after electrophoresis. Finally, clones 2# and 4# were sent to the company for sequencing to further confirm the successful construction of the plasmids.

[0066] 3) Establishment and identification of point mutation mouse models Fertilized eggs injected with sgRNA / Cas9 mRNA and donor DNA were transferred into pseudopregnant mice, resulting in the birth of F0 mice. A total of 146 F0 mice were born. The toes of the newborn F0 mice were clipped, genomic DNA was extracted, and after PCR amplification, the DNA was sent to the company for sequencing and identification. Three mice with successful point mutations were obtained, one of which died. The successfully point-mutated F0 mice were then crossed with wild-type C57 / B6 mice to obtain F1 mice. Subsequent mouse breeding strategies are as follows (…). Figure 6 ).

[0067] Since the constructed mice only had one base altered in the Zbtb20 gene (c.1787 A>G), sequencing was necessary to determine the final genotype. Because the sequence following base 1787 is CATGTTCGT, the base preceding this sequence was identified to determine the mouse genotype. Specifically, the mouse tail was first lysed using lysis buffer, and genomic DNA was extracted using isopropanol precipitation. This was then amplified by PCR, and agarose gel electrophoresis confirmed the PCR product was 419 bp in size (e.g., ...). Figure 7 A), and then send the remaining PCR products to a sequencing company for sequencing to determine the sequence. Sequence alignment will simultaneously identify the base preceding the CATGTTCGT sequence. If it is a single peak of A, it indicates a wild-type mouse. If it is a homozygous point mutation mouse, the base preceding CATGTTCGT will be a single peak of G. If there are two overlapping peaks, it indicates a heterozygous mouse. Figure 7 B).

[0068] Key sequences involved in the above process (uppercase parts are restriction endonuclease recognition sites, lowercase parts are target sequences): The top oligo sequence of sgRNA1 (SEQ ID NO.1): CACCGgcgaagatgcattgtagccc Bottom oligo (SEQ ID NO.2):AAACgggctacaatgcatcttcgcC Sequence Top oligo of sgRNA12 (SEQ ID NO.3): CACCGgaaaagatagtggttccc Bottom oligo (SEQ ID NO.4):AAACgggaaccactatcttttcC Table 1. Details of primers for the left and right homologous arms of the knock-in gene and for cutting the middle section at both ends.

[0069] II. Phenotypic Analysis of ZBTB20 Point Mutation Homozygous Mice 1) General phenotypic analysis of point mutation homozygous mice Previous studies have shown that Zbtb20 - / - Mice exhibited severe low birth weight, hypoglycemia, and early abnormal death after birth. To investigate the effects of the H596R point mutation in ZBTB20 on the organism, the effects of the point mutation on body weight and blood glucose were first observed. Under normal dietary conditions, the body weight of 3-week-old homozygous (Mut / Mut) mice was significantly lower than that of WT control mice. Figure 8 A), and was also significantly lower than that of Mut / + mice, while there was no significant difference between Mut / + mice and WT mice. Random blood glucose was also monitored, and the results showed that the random blood glucose level in 3-week-old Mut / Mut mice was significantly lower than that in the wild-type control group (A). Figure 8 B), and was also significantly lower than Mut / + mice, while random blood glucose in Mut / + mice was not significantly different from that in wild-type WT mice. In addition, it was also found that Mut / Mut mice gradually died abnormally around 3-4 weeks of age, while this phenomenon was not observed in Mut / + mice. These results suggest that the body weight, random blood glucose, and survival status of Mut / Mut mice are related to Zbtb20. - / - Similar to mice.

[0070] 2) Hippocampal developmental defects in point mutation homozygous mice To investigate the effects of the H596R point mutation in ZBTB20 on the organism, and considering the observed phenotypes in Mut / Mut mice, including low body weight, hypoglycemia, and abnormal death after birth, this further suggests that the phenotype caused by the homozygous H596R point mutation in the ZBTB20 protein may be similar to that of Zbtb20 knockout mice. Previous studies in our laboratory have shown that ZBTB20 deficiency leads to abnormal hippocampal development and structural disorder, manifested in the transformation of the CA1 region of the hippocampus into a transitional neocortical-like structure in ZBTB20-deficient mice, with the corresponding subcutaneous shift to the lateral margin of the CA3 region. Zbtb20 - / - The mouse CA1 region lacks the typical dense and homogeneous structure of the pyramidal cell layer, instead transforming into the deep and upper cortical layers of the transitional neocortex. Therefore, we continued our observation in Zbtb20... - / - This is one of the most prominent phenotypes in mice, and it is important to understand whether Mut / Mut mice also exhibit this phenotype related to Zbtb20. - / - Similar hippocampal morphological changes were observed in mice. We first performed immunohistochemistry on hippocampal sections from 3-week-old mice. The homozygous H596R point mutation of ZBTB20 did not affect the expression of ZBTB20. Figure 9 A), but interestingly, abnormal hippocampal structure was found in Mut / Mut mice, almost identical to Zbtb20. - / - Mice showed similar results, as indicated by hippocampal staining. Figure 9 B), the hippocampal cell structure of Mut / Mut mice also shows that the CA1 region lacks the classic dense and uniform pyramidal cell layer structure, and transforms into a transitional neocortical-like structure.

[0071] 3) Point mutation homozygous mice show disappearance of prolactinocytes Previous studies have shown that the absence of ZBTB20 leads to the complete loss of the mature prolactinocyte population in the anterior pituitary. Therefore, we further validated the presence of Zbtb20. - / - This prominent phenotype in mice. ZBTB20 mice were taken at 3 weeks of age. H596R Immunohistochemical results of the pituitary gland in homozygous mutant Mut / Mut mice indicated that the homozygous mutation of H596R led to pituitary PRL. + Complete absence of cell population ( Figure 10 (With Zbtb20) - / - The pituitary glands of the mice showed consistent behavior.

[0072] III. Phenotypic Analysis of ZBTB20 Point Mutation Heterozygous Mice 1) Point mutation heterozygous mice exhibit excessive growth in adulthood Primrose syndrome patients mostly exhibit age-related phenotype progression, therefore we continued to monitor their weight and length. Results showed that by 12 weeks of age, the weight and length of Mut / + mice were not significantly different from those of wild-type WT control mice. Figure 11 Surprisingly, with increasing age, the weight of Mut / + mice gradually increased significantly compared to the control mice. At 19 weeks of age, although there was no difference between the Mut / + mice and the control WT mice, they already showed a trend of weight gain. p =0.08)( Figure 11 B), at 29 weeks of age, the body weight of heterozygous Mut / + mice was significantly greater than that of control mice. Figure 11 C,F), this difference widens further at 38 weeks of age ( Figure 11 D). Surprisingly, in addition to a significant increase in body weight, we also found that the body length of Mut / + mice increased with increasing body weight. Figure 11 C', D'). When Mut / + mice were significantly heavier than control mice (at 32 weeks of age), their body length was also significantly longer than that of control mice. Figure 12 A, B). Since body length is correlated with changes in the skeleton (especially long bones), we further examined tibia length and found that the tibia of Mut / + mice was also significantly longer than that of WT mice. Figure 12 (A) This growth trend continues to increase as the mice age.

[0073] To better understand the weight growth characteristics of Mut / + mice, we monitored the weight of Mut / + mice and their littermates (WT mice) weekly and plotted growth curves. The results showed that before 21 weeks of age, there was no significant difference between Mut / + mice and their littermates (WT mice). However, with increasing age, the weight of heterozygous mice increased significantly, and at 21 weeks of age, their weight began to be significantly higher than that of their littermates (wild-type control mice), reaching a statistically significant difference. Figure 12 C). The above results suggest that Mut / + mice exhibit excessive growth in adulthood.

[0074] 2) Elevated serum IGF1 levels in point mutant heterozygous mice Does excessive growth after birth in Mut / + mice relate to the pituitary GH-IGF1 axis? Since growth hormone secretion is pulsatile, we examined the expression of IGF1, a downstream target gene of GH, in the liver. The results showed that plasma IGF1 expression was significantly upregulated. Figure 13 A), considering that IGF1 mainly originates from the liver, we measured the mRNA and protein levels of IGF1 in the liver. The results showed that both mRNA and protein levels were significantly upregulated. Figure 13 (B, C, D). This suggests that excessive growth in Mut / + mice after birth may be related to the upregulation of IGF1 expression.

[0075] 3) Point mutation heterozygotes have abnormal thyroid endocrine function To further investigate the mechanism of excessive growth after birth in Mut / + mice, considering the involvement of thyroid hormones in growth and development, and taking into account the presence of Primrose syndrome patients, we further examined thyroid function levels. We then measured plasma thyroxine levels. Interestingly, plasma thyroxine (T4) levels in Mut / + mice were significantly downregulated, but triiodothyronine (T3) levels showed no significant difference. Since T4 can be converted to T3 or reverse T3 (rT3) under the action of different deiodinases, we further measured rT3 to determine if the decrease in plasma T4 was due to abnormal conversion. The results showed no significant difference in rT3 levels between Mut / + mice and control mice. Considering that thyroxine is regulated by the hypothalamic-pituitary-thyroxine axis and feedback mechanisms, we further measured plasma TSH levels, and found that TSH was also downregulated. This suggests that the decrease in T4 levels may be due to central nervous system factors. Subsequent testing of plasma thyrotropin-releasing hormone (TRH) revealed no difference, suggesting that the decrease in thyroid hormone levels may be related to a reduction in TSH cells or their synthesis and secretion. Figure 14 ).

[0076] 4) Point mutation heterozygotes have abnormal pituitary endocrine function. The combined results above further led us to suspect that a single allele mutation in the H596R gene of ZBTB20 caused abnormal regulation of the hypothalamic-pituitary-endocrine axis. Therefore, we further examined the expression of anterior pituitary hormone-positive cells. Immunohistochemical examination of pituitary GH revealed no significant difference in the number and morphology of GH cells between WT mice and Mut / + mice. - / - In mice and ZBTB20 homozygous point mutant Mut / Mut mice, pituitary PRL-positive cells were absent, but we found no significant change in the number of PRL-positive cells in Mut / + mice. Plasma T4 and TSH levels were downregulated in Mut / + mice, but pituitary TSH cells were not reduced. We speculate that the low thyroid hormone levels in Mut / + mice may be related to abnormal TSH secretion. Figure 15 ).

[0077] 5) Analysis of cognitive function and psychobehavioral phenotypes According to current literature reports, all patients with Primrose syndrome exhibit intellectual disability, and some patients exhibit neuropsychiatric abnormalities such as anxiety, depression, schizophrenia, and autism. ZBTB20 heterozygous point mutant mice have a similar phenotype.

[0078] ①The ZBTB20 heterozygous point mutation does not affect the learning ability of mice, but it does cause spatial memory impairment.

[0079] To further investigate whether there are learning and memory impairments in Mut / + mice, we conducted a water maze test on 3-4 month old mice. The results showed that during the training phase, as the training time gradually increased, the latency to find the plateau phase in normal WT mice gradually decreased, indicating effective training. In Mut / + mice, the time to find the plateau phase gradually shortened with prolonged training, although there was a trend towards longer latency compared to WT mice (on days 7 and 9), but the difference did not reach statistical significance. Figure 16 A) indicates that the Mut / + mice have a relatively good learning ability. During the spatial exploration phase, on the first day after training ended, after the platform was removed, the experimental results showed that the Mut / + mice spent significantly less time in the quadrant containing the platform compared to the WT mice. Figure 16 C, F). To further assess whether it was related to a decrease in swimming speed, the results showed no difference in swimming speed and distance between the two groups of mice (C, F). Figure 16 The results (D, E) suggest that Mut / + mice have impaired spatial memory.

[0080] ② Long-term memory impairment in point mutant heterozygous mice To understand the long-term memory of Mut / + mice, the platform was removed again on day 10 of the training period, and the time the mice spent in the platform quadrant was assessed. The results showed that the time spent in the target quadrant and the duration of stay were significantly reduced in Mut / + mice compared to WT mice, and the number of times they entered the platform quadrant was also significantly reduced. However, it was also found that their swimming speed and distance in the target quadrant, right quadrant, and contralateral quadrant were also reduced. This suggests that the reduction in time spent in the target quadrant in Mut / + mice may be related to the reduction in swimming speed and distance. These results indicate that long-term memory is also impaired in Mut / + mice. Figure 17 ).

[0081] ③ Point mutant mice exhibit anxiety-like behavior To assess whether heterozygous point mutant mice exhibit anxiety-like behavior, we used various behavioral assessments. First, we used an open field exercise test. The results showed that, under normal circumstances, wild-type normal mice, when exploring an open field, tend to explore the peripheral areas around the "walls" due to the unfamiliarity of the new environment. However, due to their inherent curiosity, they are driven to explore the center of the open field, thus exhibiting both peripheral activity and exploration of the central area. The open field exercise trajectory and heatmap (…) Figure 18A) shows that WT mice were active in the peripheral and central areas. However, compared with WT mice, Mut / + mice showed more pronounced tracks in the periphery of the open field. Further analysis revealed that they spent more time exploring in the peripheral area, while their activity time in the central area was significantly reduced. p <0.05)( Figure 18 A, C) suggests that they exhibit anxiety-like behavior. However, the distance traveled by Mut / + mice was not significantly different from that of WT mice, suggesting that Mut / + mice do not have activity impairment. Figure 18 A, B).

[0082] To further evaluate the anxiety-like behavior of Mut / + mice, we used the black-and-white box experiment. The results showed that, under normal circumstances, wild-type mice prefer dark places and tend to avoid bright new environments, but at the same time maintain curiosity and a willingness to explore. Therefore, WT mice spent most of their time in the dark box, but also showed time spent moving around and staying in the bright box. However, Mut / + mice spent significantly more time in the dark box and significantly less time in the bright box compared to WT mice. p <0.05)( Figure 19 A, B), and the number of times they entered the open box was significantly reduced ( p <0.05)( Figure 19 C) indicates that Mut / + mice exhibit anxiety-like behavior. There was no difference in locomotor distance between the two genotypes, suggesting no difference in motor ability between the two groups of mice. Figure 19 D).

[0083] To further confirm the anxiety-like behavior of Mut / + mice, the elevated cross maze test was used for evaluation. The results showed that normal wild-type mice had a fear of being at height or suspended in the air. Therefore, normal wild-type mice exhibited activity in the dark and concealed closed arms area. However, due to their curiosity and exploratory nature, the mice were prompted to engage in activity in the open arms area. WT mice showed significantly more time spent in the closed arms area than in the open arms area. p <0.001. Mut / + mice also had significantly longer time spent with the arm closed than with the arm open, but their time spent with the arm closed was significantly longer than that of WT mice, and their time spent with the arm open was significantly shorter than that of control mice. Figure 20 (A, B) The number of times mice entered the open arm was also significantly less than that of control mice. Figure 20 C) indicates that Mut / + mice exhibit anxiety-like behavior.

[0084] Based on the results of the open field exercise experiment, black and white box experiment, and elevated cross maze experiment, it is suggested that the H596R heterozygous point mutation of ZBTB20 causes anxiety-like behavior in mice.

[0085] ④ Point mutation heterozygous mice exhibit a social novelty preference defect. During social activities in mice, compared to "familiar" mice, mice showed a greater willingness to interact with "novice" mice, exhibiting a "love of novelty" characteristic and demonstrating a memory for recognizing "familiar" versus "unfamiliar" mice. In the second phase of the three-box social experiment, when "novel" Stranger2 (S2) mice were placed in the original three empty mouse cages, we observed that normal wild-type WT mice spent more time interacting with Stranger2 mice. Figure 21 (A, B) (p<0.05), indicating that WT mice have normal novelty preference and normal memory ability. Interestingly, it was found that Mut / + mice had no difference in contact time with Stranger2 and Stranger1 mice, but their social novelty preference index was significantly lower than that of control WT mice. Figure 21 C) indicates that Mut / + mice have a poor novelty preference.

[0086] The results suggest that the H596R heterozygous point mutation in ZBTB20 results in mice exhibiting essentially normal social abilities, but unable to recognize "novel" peers, i.e., a social novelty preference deficit. We speculate that this deficit may also be caused by a social memory deficit.

[0087] 6) The overall morphology and structure of the hippocampus remained unchanged. To further investigate the mechanisms of its behavioral abnormalities, given our previous findings of hippocampal structural abnormalities in Zbtb20 knockout mice, and the similar structural changes observed in the hippocampus of ZBTB20 point mutation homozygous mice in this study, and considering the close relationship between hippocampal structure and memory, did the hippocampal structure of Mut / + mice change? We examined the hippocampal morphology of Mut / + mice using H&E staining and Nissl staining. Surprisingly, no abnormalities were found in the hippocampal morphology of Mut / + mice. Figure 22 This indicates that the heterozygous site mutation of ZBTB20 did not affect the gross structure of the hippocampus.

[0088] 7) Point mutation heterozygous mice do not affect the nucleoplasmic distribution in ZBTB20 hippocampal neurons. Considering ZBTB20 as a transcription factor, we investigated whether the abnormal phenotypes observed in Mut / + mice were related to alterations in its nuclear localization. Therefore, we used immunohistochemistry to further investigate ZBTB20 expression. The results showed that the nucleoplasmic distribution of ZBTB20 in hippocampal neurons of Mut / + mice was not altered. Figure 23 ).

[0089] 8) Dendritic development disorder in CA1 region of hippocampus To further validate the single-cell sequencing results, we performed Golgi staining on neurons in the brain tissues of Mut / + mice and their littermate wild-type control mice, and conducted Sholl analysis on neurons in the CA1 region of the hippocampus. Figure 24 As shown, at a distance of 50-60 μm from the cell body, the number of intersections between dendrites and concentric circles in Mut / + mice was significantly lower than that in wild-type mice. p <0.05). Simultaneously, a comparison of dendritic spine density in the CA1 region of the hippocampus between the two groups of mice revealed that, although there was no difference in total dendritic spine density between the two groups, the density of mature mushroom-shaped dendritic spines in Mut / + mice decreased by approximately 30% compared to wild-type mice. p <0.05) Figure 25 These results indicate that Mut / + mice have reduced dendritic branching in the CA1 region of the hippocampus and decreased density of mature mushroom-shaped dendritic spines.

[0090] 9) Transcriptomic analysis of the hippocampus in point mutation heterozygous mice Mut / + mice exhibited behavioral abnormalities, but no abnormalities were observed in hippocampal morphology. We then used high-throughput transcriptomics to analyze the gene expression profile of hippocampal tissue to further explore its potential mechanisms. Based on the sequencing results, GO (Gene Ontology) differential gene enrichment analysis was first performed on the hippocampal tissues of Mut / + and wild-type WT control mice. The top 20 differentially enriched genes included: axonogenesis, synapse organization, regulation of membrane potential, synapse assembly, regulation of ion transmembrane transport, presynapse assembly, regulation of synapse organization, cell junction assembly, regulation of synapse structure or activity, calcium ion transmembrane transport, regulation of heart contraction, axon guidance, neuron projection guidance, regulation of blood circulation, regulation of neuron death, and the Wnt signaling pathway. pathway), Wnt intercellular signaling, heart contraction, and reproductive structure development. Figure 26 Based on the above enrichment analysis, the main enriched pathways are related to axons.

[0091] Further enrichment analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway revealed enrichment primarily in oxidative phosphorylation, Alzheimer's disease (AD), glutamatergic synapses, neurodegeneration, reactive oxygen species, cholinergic synapses, calcium signaling pathways, thyroid hormone signaling pathways, long-term potentiation (LTP) cAMP signaling pathways, and dopaminergic synapses. Figure 27 The above analysis of GO enrichment pathways and KEGG signaling pathways suggests that the behavioral abnormalities in Mut / + mice may be closely related to oxidative phosphorylation, synaptic function, and calcium ion signaling pathways.

[0092] Next, we performed GSEA (Gene Set Enrichment Analysis). Based on the NES enrichment scores, the main upregulated pathways included negative regulation of interleukin-6 production, gastric motility, geramide-1 phosphate transport, and the immune response regulating cell surface receptor signaling pathway. Downregulated pathways included the antigen receptor mediated signaling pathway, muscle cell development, granulocyte colony-stimulating factor production, growth-related heart morphogenesis, positive regulation of skeletal muscle fiber development, alanine catabolic process, electron transport chain, mitochondrial electron transport NADH to ubiquinone, cytoplasmic translation, and the NADH dehydrogenase complex. The pathways involved include assmenbly, myotube cell development, oxidative phosphorylation, positive regulation of muscle tissue development, and ATP synthesis coupled electron transport. Among these pathways, we particularly note those closely related to mitochondrial respiration, such as the electron transport chain enriched in these pathways, mitochondrial electron transport from NADH to ubiquinone, the NADH dehydrogenase complex, oxidative phosphorylation, and ATP synthesis coupled electron transport. Figure 28This strongly suggests that it may be highly related to mitochondrial respiratory metabolism.

[0093] Further analysis using GSEA (Gene Set Enrichment Analysis) revealed that in both the Wikipathways database and the Reactome pathway database... Figure 29 In this study, these downregulated genes can be enriched in the mitochondrial respiratory chain or oxidative phosphorylation pathway.

[0094] Based on the RNA-seq bioinformatics analysis results of Shanghai horse tissue, we strongly suggest that the behavioral abnormalities in mice caused by the H596R point mutation of ZBTB20 may be related to mitochondrial cellular respiratory metabolism. 10) Hippocampal cell clusters and characteristics in mice In this study, a total of 7 hippocampal tissue samples were sequenced into single cells (3 WT mice and 4 Mut / + mice). After quality control (QC) to remove low-quality cells (cells with a high proportion of mitochondrial genes and abnormal gene counts), transcriptional information from 82,028 single cells was obtained. Data visualization was performed using two dimensionality reduction clustering methods: t-distributed stochastic neighbor embedding (t-SNE) probabilistic algorithm and uniform manifold approximation and projection (UMAP). Both algorithms can cluster cells into 32 cell clusters. Figure 30 , Figure 31 ).

[0095] Next, specific marker genes for hippocampal-related cells were obtained based on literature reports. Based on these marker genes, the cells were grouped and annotated into 22 cell populations: Cluster 1: Astrocytes (mainly expressing Slc1a3, Slc1a32); Cluster 4: CA1 Pyramidal cells (CA1 Pyr; mainly expressing Man1a, Ryr3, Homer1); Cluster 10: CA2 Pyramidal cells (CA2 Pyr); Cluster 8: CA3 Pyramidal cells (CA3 Pyr; mainly expressing Grik4, Gabbr2, Erc2); Cluster 15: Cajal-Retzius cells (CR; mainly expressing Reln, Trp73); Cluster 5: Endothelial cells (Endo; mainly expressing Flt1); and Cluster 6: GABAergic neurons (GABAergic neurons). Neuron (GABA, mainly expressing Elavl2), Granular Cells (Granule, mainly expressing prox1, Trpc6) in Cluster 0, Intermediate progenitor cells (IPCs) in Cluster 20, Interneurons in Cluster 16, Microglia (Microglia1, mainly expressing C1qa) in Cluster 7, Neuroblasts (Neuroblasts, mainly expressing Nnat) in Cluster 16, Neuroblasts 2 in Cluster 9, Newly formed oligodendrocytes (NFOL) in Cluster 19, Oligodendrocyte 1 (OL1, mainly expressing Plp1, Mag, Mbp) in Cluster 25, Oligodendrocyte 2 (OL1, OL1, mainly expressing Plp1, Mag, Mbp). 2,OL2; mainly expressing Flt1, Plp1, Mag, Mbp), and oligodendrocyte 3 (OL3) of cluster 27;The main expression groups are C1qa, Plp1, Mag, and Mbp; oligodendrocyte progenitor cells (OPCs) in cluster 13; pericytes in cluster 14; vascular leptomeningeal cells 1 (VLMC1) in cluster 11; vascular leptomeningeal cells 1 (VLMC2) in cluster 18; and an unknown group (Unknown, unk) in cluster 3 that could not be annotated. Figure 32 , Figure 33 ).

[0096] Based on the annotation and grouping of these cell clusters, the top 10 predominant cell groups in the hippocampus are granular cells, astrocytes, oligodendrocytes 1, an unannotated unknown group, CA1 pyramidal cells, endothelial cells, γ-aminobutyric acid (GABAergic) neurons, microglia 1, CA3 pyramidal cells, and neuroblasts 2 (…). Figure 34 , Figure 35 ).

[0097] Cell subpopulation annotation based on hippocampal marker genes yielded 22 cell subpopulations, including astrocytes, CA1 pyramidal cells, CA2 pyramidal cells, CA3 pyramidal cells, endothelial cells, GABAergic neurons, granule cells, intermediate progenitor neurons, oligodendrocytes 1, microglia 1, microglia 2, microglia 3, neuroblasts 2, and unannotated unknown subpopulations.

[0098] 11) Changes in hippocampal cell subsets in point mutant mice Next, we conducted a cell subset analysis comparing the Mut / + and WT mice of the two genotypes. Figure 36Surprisingly, the most significant changes in both genotypes of mice were observed in two main cell populations: CA1 pyramidal cells and an unknown cell type, unk cells. The CA1 pyramidal cell population was significantly decreased (8.28% vs 5.86%), while the unk cell population was significantly increased (6.98% vs 10.53%). Other cell subpopulations also showed changes, such as decreased CA3 pyramidal cells (4.26% vs 3.58%), GABA cells (4.56% vs 3.88%), neuroblasts (3.59% vs 3.29%), and oligodendrocytes (11.45% vs 10.56%), while increased granular cells (19.85% vs 21.49%) and CR cells (0.58% vs 0.77%). Most of these cell subpopulations showed cell reduction, suggesting that this reduction may be the main reason for the behavioral abnormalities in Mut / + mice caused by the ZBTB20 heterozygous point mutation.

[0099] 12) Downregulation of glutamatergic and synaptic transmission pathways in CA1 pyramidal cells of hippocampal neurons in point mutant mice Previous literature has shown that ZBTB20 is involved in the development of the CA1 cell population. Among the cell subpopulations with significantly reduced proportions, we first focused on the CA1 cell population. GSEA analysis of CA1 cell subpopulations from two mouse genotypes revealed that, among the downregulated signaling pathways, the most significant enrichment was in pathways related to synapse and excitatory neurotransmitter transmission, such as the positive regulation of synaptic transmission glutamatergic, synapse maturation, the glutamate receptor signaling pathway, and the regulation of neurotransmitter receptor activity. Figure 37 (38), strongly suggesting possible changes in synapse formation and glutamate neurotransmitter transmission in CA1 pyramidal neurons.

[0100] 13) Downregulation of synaptic transmission and synaptic plasticity-related pathways in CA3 pyramidal cells of hippocampal neurons in point mutant mice. Since CA3 pyramidal neurons are involved in the hippocampal memory circuit, and the proportion of CA3 pyramidal neuron cells in the subpopulation was also decreased, we further analyzed the CA3 cell subpopulation. We found that the most significant downregulations were in the regulation of neurotransmitter receptor activity, regulation of excitatory postsynaptic potential, transmission of nerve impulse, positive regulation of excitatory postsynaptic potential, regulation of neuronal synaptic plasticity, and regulation of long-term neuronal synaptic plasticity. Figure 39 ,40).

[0101] 14) Downregulation of neuronal projection and synaptic excitability-related pathways in hippocampal granule cells of point-mutated mice Granular neurons in the DG region participate in the hippocampal trisynaptic circuit. Further analysis of granular cell subsets using GSEA enrichment revealed downregulation of pathways involved in neuronal projection or postsynaptic potentials, such as neuron projection extension involved in neuron projection guidance, modulation of excitatory postsynaptic potential, olfactory interneuron differentiation, cannabinoid signaling pathway, adenylate cyclase inhibiting G protein-coupled acetylcholine receptor signaling pathway, and endocannabinoid signaling pathway. Figure 41 ,42).

[0102] Based on the GSEA analysis results of CA1 and CA3 pyramidal neurons and granule cells, we suggest that heterozygous point mutations downregulate excitatory neuronal synaptic transmission pathways in the hippocampal trisynaptic circuit. Therefore, we hypothesize that point-mutant mice may have impaired excitatory transmission in hippocampal circuit neurons. Further analysis of single-cell sequencing results will provide directions and clues for future research.

[0103] 15) ZBTB20 H596R Mutation leads to impaired hematoxylinization modification of ZBTB20 We further tested ZBTB20. H596R The effect of mutations on hematoxylinization modification of ZBTB20. We will investigate the effect of mutations on hematoxylinization modification of ZBTB20. H596R Mutant plasmids were transfected into HEK293A cells, and K330R, K371R, and K330 / K371R (2KR) plasmids with ZBTB20 hematoxylinization modification sites were transfected as controls. Changes in the ZBTB20 hematoxylinization modification level in these mutants were analyzed. Results showed that hematoxylinized ZBTB20 protein bands were detected in transfected wild-type ZBTB20 protein, while bands in transfected wild-type ZBTB20 protein were significantly reduced. H596R In mutant cells, the level of hematoxylinization modification in ZBTB20 was significantly lower than that in wild-type ZBTB20. Interestingly, transfection with ZBTB20 containing the hematoxylinization modification site mutation... K330R and ZBTB20 K330R / K371R In the (2KR) mutant cells, no hematoxylinization-modified protein bands were detected. These results suggest that hematoxylinization is a common post-translational modification of ZBTB20. H596R The mutation reduces its hematoxylinization modification level. We further clarified the ZBTB20 mutation through IP experiments. H596R Does the mutant protein affect its hematoxylinization modification level? We transfected exogenous Flag-SUMO and UBC9 (SUMO-E2 coupled enzymes) into HEK293A cells, and then transfected ZBTB20 wild-type and H596R mutants, respectively. IP was performed using anti-Flag antibody, and Western blot was used to detect changes in hematoxylinization modification of ZBTB20. The results showed that compared with wild-type ZBTB20, ZBTB20… H596R The mutant's ability to bind the SUMO protein was significantly lower than that of the wild-type ZBTB20. In conclusion, ZBTB20... H596R The mutation leads to impaired hematoxylinization modification of ZBTB20 ( Figure 43 ).

[0104] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A non-human animal model of Primrose syndrome, characterized in that, That Zbtb20 The gene contains a c.1787 A>G point mutation, which leads to a p.H596R amino acid substitution in its encoded protein.

2. The non-human animal model of Primrose syndrome according to claim 1, characterized in that, The animals mentioned include mice.

3. The method for constructing a non-human animal model of Primrose syndrome as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Design and prepare targeted Zbtb20 sgRNA in the intron regions flanking exon 14 of a gene; S2. Construct a homologous recombination targeting vector containing the c.1787 A>G point mutation site; S3. Inject the Cas9 mRNA, the sgRNA prepared in step S1, and the targeting vector constructed in step S2 into the fertilized egg. S4. The injected fertilized eggs were transferred into pseudopregnant mice to obtain F0 generation mice. S5. Genotyping of F0 generation mice was performed to screen out individuals carrying the c.1787 A>G point mutation.

4. The construction method according to claim 3, characterized in that, In step S1, the sgRNA is screened for activity using the luciferase reporter gene method, and sgRNAs with high cleavage activity are selected for subsequent experiments.

5. The construction method according to claim 3, characterized in that, In step S2, the homologous recombination targeting vector contains... Zbtb20 The left and right homologous arms flanking the gene target site, and the sequence containing the c.1787A>G point mutation located between the homologous arms.

6. The construction method according to claim 3, characterized in that, In step S5, the genotype identification includes: extracting mouse genomic DNA, amplifying the Zbtb20 gene fragment containing the c.1787 site by PCR, sequencing the PCR product, and determining the genotype by analyzing the base type before the CATGTTCGT sequence in the sequencing results.

7. The application of the non-human animal model of Primrose syndrome as described in any one of claims 1 to 2 in the study of the pathophysiological mechanism of Primrose syndrome.

8. The use of the non-human animal model of Primrose syndrome as described in any one of claims 1 to 2 in the development of products for diagnosing Primrose syndrome.

9. The use of the non-human animal model of Primrose syndrome as described in any one of claims 1 to 2 in screening drugs for the treatment of Primrose syndrome.

10. The use of the non-human animal model of Primrose syndrome as described in any one of claims 1 to 2 in the development of new drugs for the treatment of Primrose syndrome.

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