Inpp5e gene conditional knockout animal model construction method and application
The Inpp5e gene conditional knockout mouse model was constructed using the CRISPR/Cas9 system, which solved the problem of insufficient research on the Inpp5e gene in existing technologies and enabled in-depth research and drug development for neurodevelopmental related diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-27
AI Technical Summary
Current research on the Inpp5e gene is limited, and there is a lack of effective conditional gene knockout animal models for studying the etiology and pathogenesis of neurodevelopmental related diseases.
sgRNA was designed using the CRISPR/Cas9 system and a targeting vector was constructed. The Inpp5e gene conditional knockout mouse model was established by microinjecting sgRNA and Donor fragment into mouse zygotes.
This provides a simple and easy-to-use method for studying the etiology and pathogenesis of various neurodevelopmental disorders and for developing drugs for these diseases.
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Figure CN121737217A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biomedicine, and particularly to a method for constructing an Inpp5e gene conditional knockout animal model and application of the Inpp5e gene conditional knockout animal model in neural development related diseases. BACKGROUND
[0002] Inositol polyphosphate-5-phosphatase E (Inpp5e) gene is located on human chromosome 9q34.3, contains 10 exons, and has a base number of 3984 bp. The gene encodes a protein INPP5E composed of 644 amino acids and having a molecular weight of 72 kDa, and belongs to the 5-phosphatase family. The gene is a key enzyme in phosphatidylinositol metabolism and has various important biological functions, and is a key signal molecule in many intracellular signal transduction pathways. Studies have shown that the Inpp5e gene plays an important role in embryonic neural development.
[0003] However, the related research on Inpp5e is still limited. The construction of a gene conditional knockout animal model can provide a good research model for the study of various neural development related diseases, especially the etiology and pathogenesis of the gene involved in the metabolic pathway and neural development defects, and can provide services for the further development of drugs for such diseases. SUMMARY
[0004] To overcome the defects of the prior art, the present application solves the technical problem of providing a method for constructing an Inpp5e gene conditional knockout animal model, which provides a good basis for the etiology and pathogenesis of the gene involved in the metabolic pathway and neural development defects.
[0005] The technical solution of the present application is that the method for constructing an Inpp5e gene conditional knockout animal model comprises the following steps: (1) sgRNA design: Inpp5e cko usgRNA1 is shown as SEQ ID NO. 1, Inpp5e cko usgRNA2 is shown as SEQ ID NO. 2, Inpp5e cko dsgRNA1 is shown as SEQ ID NO. 3, and Inpp5e cko dsgRNA2 is shown as SEQ ID NO. 4; (2) Targeting vector construction: The targeting vector includes four fragments, a 1986 bp left homologous arm LHA, an 871 bp middle insertion fragment loxp, a 2079 bp right homologous arm RHA, and a 2659 bp plasmid backbone pUC19L; the LHA and RHA are obtained by PCR amplification using mouse tail lysis fluid of C57BL / 6J wild mice as a template, and the loxp is obtained by PCR amplification using a Blunt-inpp5e loxp plasmid as a template; the plasmid backbone uses a pUC19L linearized vector; the four fragments LHA, loxp, RHA and the pUC19L linearized vector obtained by purification and recovery are spliced by a one-step seamless cloning and assembly kit to obtain the targeting vector pUC19L-Inpp5e CKO Targeting Vector; endotoxin removal is performed using a large-scale plasmid extraction kit, and the large-scale plasmid obtained is digested with a restriction endonuclease PvuI, and a 6.7 Kb fragment is recovered as a donor for microinjection; (3) Microinjecting the Inpp5e cko usgRNA1, the Inpp5e cko dsgRNA1 and the donor into mouse zygotes to obtain an Inpp5e gene conditional knockout mouse.
[0006] The beneficial technical effects of the present application are as follows: The present application uses a CRISPR / Cas9 system to construct an Inpp5e gene conditional knockout mouse model, and the method is simple and easy to operate; the mouse model can be used for research on various neural development-related diseases, especially for providing a good research model for studying the causes and pathogenesis of genes and neural development defects related to the metabolic pathway of the gene, and for further developing drugs for such diseases.
[0007] The application of the Inpp5e gene conditional knockout animal model for non-disease diagnosis and treatment purposes is also provided, and the application includes: using the Inpp5e gene conditional knockout animal model obtained by the construction method for research on neural development-related diseases. BRIEF DESCRIPTION OF DRAWINGS
[0008] Figure 1 It is shown as a pUC19L-Inpp5e CKO Targeting Vector map.
[0009] Figure 2 It is shown as a Blunt-Inpp5e Loxp colony identification gel electrophoresis map.
[0010] Figure 3The image shown is a gel image of colony identification using the pUC19L-Inpp5e CKO Targeting Vector.
[0011] Figure 4 The results are shown as pUC19L-Inpp5e CKO Targeting Vector-4# sequencing results.
[0012] Figure 5 The image shown is an electrophoresis diagram of pUC19L-Inpp5e CKO Targeting Vector-4# after enzyme digestion.
[0013] Figure 6 The results show the Inpp5e-cKO-usgRNA1 cleavage activity assay.
[0014] Figure 7 The results show the Inpp5e-cKO-dsgRNA1 cleavage activity assay.
[0015] Figure 8 The image shows a gel electrophoresis diagram of the coarse screening genotype identification of Inpp5e-cKO F0 mice 5393♂. P: mouse tail lysis buffer mixture; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water.
[0016] Figure 9 The image shows a random insertion identification gel electrophoresis diagram of Inpp5e-cKO F0 mouse 5393♂. P: tail lysis buffer of mouse number 5555; N (negative control): tail lysis buffer of mouse C57; B (blank control): sterile water.
[0017] Figure 10 The image shows a gel electrophoresis diagram of the primers used to identify Inpp5e-cKO F0 mice (5393♂). P: mouse tail lysis buffer mixture; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water.
[0018] Figure 11 The image shows a gel electrophoresis plot of primers used to identify the linkers of the PCR product from Inpp5e-cKO F0 mice (5393♂). The numbers represent the linker numbers.
[0019] Figure 12 The result displayed is the upstream sequencing result for #5393.
[0020] Figure 13 The result shown is the downstream sequencing result for 5393#.
[0021] Figure 14 The image shown is a gel electrophoresis diagram of F1 generation mice for preliminary screening and identification.
[0022] Figure 15The image shown is a gel electrophoresis diagram of F1 generation mice randomly inserted for identification.
[0023] Figure 16 The image shown is a gel electrophoresis diagram of F1 generation mice.
[0024] Figure 17 The image shows gel electrophoresis images of large fragments identified in mice 8319♀ and 8645♂.
[0025] Figure 18 The results are shown as sequencing results for 8319♀.
[0026] Figure 19 The sequencing result is shown as 8645♂.
[0027] Figure 20 The results show sequencing results for 9097♀.
[0028] Figure 21 The result is shown as 9100♀ sequencing results.
[0029] Figure 22 The diagram shows the creation of a conditional gene knockout. Detailed Implementation
[0030] This method for constructing an Inpp5e gene conditional knockout animal model includes the following steps: (1) sgRNA design: Inpp5e cko usgRNA1 is shown in SEQ ID NO.1, Inpp5e cko usgRNA2 is shown in SEQ ID NO.2, Inpp5e cko dsgRNA1 is shown in SEQ ID NO.3, and Inpp5e cko dsgRNA2 is shown in SEQ ID NO.4; (2) Construction of the targeting vector: The targeting vector consists of four fragments: a 1986 bp left homologous arm LHA, an 871 bp middle insert loxp, a 2079 bp right homologous arm RHA, and a 2659 bp plasmid backbone pUC19L. LHA and RHA were obtained by PCR amplification using tail lysate from C57BL / 6J wild-type mice as templates, and loxp was obtained by PCR amplification using the Blunt-inpp5e loxp plasmid as a template. The plasmid backbone was linearized using pUC19L. The four purified and recovered fragments LHA, loxp, RHA, and pUC19L linearized vector were seamlessly spliced in one step using the pEASY®-Basic Seamless Cloning and Assembly Kit to obtain the targeting vector pUC19L-Inpp5e CKO Targeting Vector. Endotoxin-free large-scale extraction was performed using a large-scale plasmid extraction kit. The obtained large-scale plasmid was digested with restriction endonuI and recovered. Kb fragments were used as donors for microinjection; (3) Inpp5e cko usgRNA1, Inpp5e cko dsgRNA1 and Donor were microinjected into mouse zygotes to obtain Inpp5e gene conditional knockout mice.
[0031] The beneficial technical effects of the present invention are as follows: The present invention provides a simple and easy method for constructing a conditional knockout mouse model of the Inpp5e gene using the CRISPR / Cas9 system. This mouse model can be used to study a variety of neurodevelopmental diseases, especially providing a good research model for the study of the etiology and pathogenesis of gene and neurodevelopmental defects in the metabolic pathways involved in this gene, and serving the further development of drugs for such diseases.
[0032] Preferably, in step (1), the concentrations of Inpp5e cko usgRNA1, Inpp5e cko usgRNA2, Inpp5ecko dsgRNA1, and Inpp5e cko dsgRNA2 are 3269 ng / μL, 3104 ng / μL, 3069 ng / μL, and 3200 ng / μL, respectively.
[0033] Preferably, step (2) further includes identification of the target vector bacterial culture: the Blunt-Inpp5e loxp plasmid is constructed by amplifying the Loxp fragment by high-fidelity polymerase PCR, which contains two Loxp fragments and exon7 and exon8, and after purification and recovery, it is ligated into the Blunt blunt-end vector; the Blunt-Inpp5e loxp bacterial culture is identified by gel electrophoresis. The bacterial culture containing the pUC19L-Inpp5e CKO Targeting Vector plasmid was identified using three pairs of primers, yielding the following products: 740 bp Inpp5e-JDF1+Inpp5e-JDR1, 736 bp Inpp5e-JDF2+Inpp5e-JDR2, and 713 bp Inpp5e-JDF3+Inpp5e-JDR3. The bacterial culture was then identified by agarose gel electrophoresis.
[0034] Preferably, step (2) further includes sequencing identification of the targeting vector: sequencing the bacterial culture and sequencing the 4936 bp LHA-Loxp-RHA fragment.
[0035] Preferably, in step (3), embryo transfer is performed on the same day as microinjection, and the mice are born 21 days after the transfer.
[0036] Preferably, the method further includes step (4), where the tail of the mouse is clipped about 10 days after birth for identification; and one Founder is obtained by PCR and sequencing identification.
[0037] Preferably, in step (4), the identification primer sequences are: Inpp5eCKOcushaiF as shown in SEQ ID NO.5, Inpp5eCKOcushaiR as shown in SEQ ID NO.6, Inpp5eckoCF as shown in SEQ ID NO.7, Inpp5eckoCR as shown in SEQ ID NO.8, Inpp5ecko-RIR3 as shown in SEQ ID NO.9, Inpp5ecko-RIF3 as shown in SEQ ID NO.10, Inpp5e cko uF as shown in SEQ ID NO.11, Inpp5e cko uR as shown in SEQ ID NO.12, Inpp5e cko dF as shown in SEQ ID NO.13, and Inpp5e cko dR as shown in SEQ ID NO.14.
[0038] Preferably, in step (4), in PCR, Inpp5eCKOcushaiF+Inpp5eCKOcushaiR, Inpp5e-cko-RI-F3+Inpp5e-cko-RI-R3 adopt a three-step method using Kangwei Century's mix; Inpp5eckoCF+Inpp5eckocushaiR, Inpp5eckocushaiF+Inpp5eckoCR, Inpp5eckoCF+Inpp5eckoCR adopt a two-step method using TOYOBO KOD One PCR Master Mix-Blue.
[0039] Preferably, the method further includes mating and propagation. After the positive Founder mice reach sexual maturity, they are mated with C57BL / 6J mice in the same cage. Four F1 generation positive mice are obtained after identification, numbered 8319♀, 8645♂, 9097♀, and 9100♀. Inpp5e CKO CF + Inpp5e CKO CR is sequenced using Inpp5e cko uF and Inpp5e cko dR. The large fragment sequencing results of number 8319♀ show that there are overlapping peaks at two Loxp positions, indicating the presence of two Loxp fragments. 8319♀ does not contain random insertions. Amplification is performed using primers Inpp5e cko uF + Inpp5e cko dR, and 1475 The amplified fragment of 1475 bp was ligated into the Blunt vector, and positive clones were selected for sequencing. The sequencing results showed that it contained two Loxp sequences of the target sequence. The sequencing results of the large fragment of number 8645♂ showed that there were overlapping peaks at the positions of the two Loxp sequences, and the Loxp sequences were also present in the fragments of LA and RA. 9097♀ and 9100♀ did not contain random insertions. They were amplified using primers Inpp5e cko uF+ Inpp5e cko dR, and the amplified fragment of 1475 bp was ligated into the Blunt vector. Positive clones were selected for sequencing, and the sequencing results showed that it contained two Loxp sequences of the target sequence. Therefore, the sequencing results showed that 8319♀, 8645♂, 9097♀, and 9100♀ were all positive F1 generation mice.
[0040] It also provides applications for Inpp5e gene conditional knockout animal models not intended for disease diagnosis and treatment, including using Inpp5e gene conditional knockout animal models obtained by the construction method to study neurodevelopmental related diseases.
[0041] The embodiments of the present invention will be described in detail below.
[0042] 1. sgRNA design and vector construction Table 1. sgRNA sequence information and transcript concentration.
[0043] 2. Construction of the target carrier The targeting vector comprises four fragments: the left homologous arm LHA (1986 bp), the middle insert fragment loxp (871 bp), the right homologous arm RHA (2079 bp), and the plasmid backbone pUC19L (2659 bp). Fragments LHA and RHA were obtained by PCR amplification using tail lysates from C57BL / 6J wild-type mice as templates, while fragment loxp was obtained by PCR amplification using the Blunt-inpp5e loxp plasmid (constructed by Viton Technologies) as a template. The plasmid backbone was constructed using the pUC19L linearized vector (preserved by Viton Technologies). The purified and recovered fragments LHA, loxp, RHA, and the pUC19L linearized vector were seamlessly assembled in a one-step process using the pEASY®-Basic Seamless Cloning and Assembly Kit (CU201-02, All-Gold), successfully obtaining the targeting vector pUC19L-Inpp5e CKO Targeting Vector. The targeting vector map is shown below. Figure 1 .
[0044] 2.1 Identification of Bacterial Fluids for Targeting Vectors The Blunt-Inpp5e loxp plasmid was constructed by high-fidelity polymerase PCR amplification of the Loxp fragment (containing two Loxp fragments and exon7, exon8), purified, and ligated into the Blunt blunt-end vector (pEASY®-Blunt Cloning Kit, CB101). Gel electrophoresis images of Blunt-Inpp5e loxp bacterial culture identification are shown below. Figure 2 .
[0045] The pUC19L-Inpp5e CKO Targeting Vector plasmid bacterial suspension was identified using three primer pairs: Inpp5e-JDF1+Inpp5e-JDR1 (740 bp), Inpp5e-JDF2+Inpp5e-JDR2 (736 bp), and Inpp5e-JDF3+Inpp5e-JDR3 (713 bp). Agarose gel electrophoresis images of the bacterial suspension are shown below. Figure 3 .
[0046] 2.2 Sequencing and Identification of Targeting Vectors The bacterial culture #4 was sent for sequencing, and the LHA-Loxp-RHA (4936 bp) fragment was sequenced. The sequencing results showed that the sequence alignment of the bacterial culture #4 was completely correct. The sequencing results are shown below. Figure 4 .
[0047] 2.3 Preparation of Injection Donor The correctly sequenced pUC19L-Inpp5e CKO Targeting Vector-4# was subjected to endotoxin-free plasmid extraction using a large-scale plasmid extraction kit (TransGold Endotoxin-Free Plasmid Large-Scale Extraction Kit; EM123). The obtained large-scale plasmid was digested with the restriction endonuclease PvuI, and the 6.7 kb fragment was recovered as the donor for microinjection. The gel electrophoresis image of the donor preparation is shown below. Figure 5 .
[0048] 3. Microinjection The sgRNA cleavage activity assay showed that Inpp5e cko usgRNA1 and Inpp5e cko dsgRNA1 had high cleavage activity, so these two sgRNAs were used for subsequent injection experiments. Figure 6 The results show the Inpp5e-cKO-usgRNA1 cleavage activity assay. Figure 7 The results show the Inpp5e-cKO-dsgRNA1 cleavage activity assay.
[0049] Microinjection began on May 20, 2024. Injection information is shown in the table below.
[0050] Table 2
[0051] 4. Mice birth and testing Embryo transfer was performed on the day of microinjection, and mice were born 21 days later. Approximately 10 days after birth, the mice's tails were clipped for identification. PCR and sequencing identified one Founder mouse, designated 5393♂.
[0052] 4.1 Identification Methods The primer sequences for identification are shown in Table 3.
[0053] Table 3
[0054] PCR conditions Inpp5eCKOcushaiF+Inpp5eCKOcushaiR: Three-step method, using Kangwei Century's mix, see Table 4.
[0055] Table 4
[0056] Inpp5e-cko-RI-F3+Inpp5e-cko-RI-R3: Three-step method, using Kangwei Century's mix, see Table 5.
[0057] Table 5
[0058] Inpp5eckoCF+Inpp5eckocushaiR and Inpp5eckocushaiF+Inpp5eckoCR: Two-step method using TOYOBO KOD One PCR Master Mix-Blue, see Table 6.
[0059] Table 6
[0060] Inpp5eckoCF+Inpp5eckoCR: Two-step method using TOYOBO KOD One PCR Master Mix-Blue, see Table 7.
[0061] Table 7
[0062] The PCR detection system is shown in Tables 8 and 9.
[0063] Table 8
[0064] Table 9
[0065] 4.2 PCR identification of Founder electrophoresis diagram Figure 8 Displayed as Inpp5e -cKO F0 mice 5393♂ coarse screening genotype identification gel electrophoresis image. P: mouse tail lysis buffer mixture; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water. Figure 8 F0 (5393♂) is a preliminary positive mouse and will be further identified.
[0066] Figure 9 Displayed as Inpp5e -cKO F0 mice 5393♂ randomly inserted identification gel electrophoresis image. P: tail lysis buffer of mouse number 5555; N (negative control): tail lysis buffer of mouse C57; B (blank control): sterile water. Figure 9 The result showed that 5393♂ did not contain random insertions, and further identification was performed using detection primers.
[0067] Figure 10 Displayed as Inpp5e -cKO F0 mouse 5393♂ identification primer gel electrophoresis image P: mouse tail lysis buffer mixture; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water. Figure 10 The results showed that mouse 5393 was a cKO-positive mouse, and further sequencing was performed to test this mouse.
[0068] 4.3 Sequencing results of Founder generation mice Amplification was performed using Inpp5e cko uF + Inpp5e cko dR, and the amplified fragment (1475 bp) was ligated into the Blunt vector. Positive clones were selected and sequenced using primers Inpp5e cko uF + Inpp5e cko dF. Sequencing results showed that 5393 contained two Loxp sequences, indicating a positive mouse.
[0069] 4.4 Information on positive Founder mice is shown in Table 10.
[0070] Table 10
[0071] 5. Mating and propagation Once the positive Founder mice reached sexual maturity, they were mated together with C57BL / 6J mice. The mating and birth details of the mice are shown in Table 11, and a total of 4 F1 generation positive mice were obtained.
[0072] Table 11
[0073] 5.1 F1 generation mouse detection Founder mouse 5393 (♂) produced 38 offspring, and 4 F1 generation positive mice were obtained after identification, numbered 8319♀, 8645♂, 9097♀, and 9100♀.
[0074] Figure 14 The results show that mice numbered 8319♀, 8645♂, 9097♀, and 9100♀ are preliminarily screened positive F1 generation mice and will be further identified. P (positive control): 5555 mouse tail lysis buffer; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water.
[0075] Figure 15 The identification results showed that the four positive mice initially screened, numbered 8319♀, 8645♂, 9097♀, and 9100♀, did not contain random insertions, which was consistent with the test results of the parent 5393.
[0076] Figure 16 The results showed that four F1 generation mice, 8319♀, 8645♂, 9097♀, and 9100♀, were cKO-positive. Further sequencing was performed on these four mice. P (positive control): 5393 mouse tail lysis buffer; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water.
[0077] Figure 17The image shows large fragment identification gel electrophoresis images of mice 8319♀ and 8645♂. P (positive control): 5393 mouse tail lysis buffer; N (negative control): C57 mouse tail lysis buffer; B (blank control): sterile water.
[0078] 5.2 Sequencing Identification Sequencing primers: PCR amplification of large fragments (Inpp5e CKO CF + Inpp5e CKO CR) was performed using Inpp5e cko uF and Inpp5e cko dR. The sequencing results of fragment 8319♀ showed overlapping peaks at two Loxp positions, indicating the presence of two Loxp fragments. Since 8319 did not contain random insertions, we amplified it using primers Inpp5e cko uF and Inpp5ecko dR. The amplified fragment (1475 bp) was ligated into the Blunt vector, and positive clones were selected for sequencing. The sequencing results showed the presence of the target sequence at two Loxp positions. The sequencing results of fragment 8645♂ showed overlapping peaks at two Loxp positions, and the Loxp sequence was present in both the LA and RA fragments. Since mice 9097♀ and 9100♀ did not contain random insertions, they were amplified using primers Inpp5e cko uF and Inpp5e cko dR. The amplified fragment (1475 bp) was ligated into the Blunt vector, and positive clones were selected for sequencing. Sequencing results showed that two Loxp clones contained the target sequence. Therefore, sequencing results showed that mice 8319♀, 8645♂, 9097♀, and 9100♀ were all positive F1 generation mice. See [link to sequencing results] for details. Figure 18 , Figure 19 , Figure 20 , Figure 21 .
[0079] 5.3 Information on F1 generation mice is shown in Table 12.
[0080] Table 12
[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. A method for constructing an animal model of conditional knockout of the Inpp5e gene, characterized by: It includes the following steps: (1) sgRNA design: Inpp5e cko usgRNA1 is shown in SEQ ID NO.1, Inpp5e cko usgRNA2 is shown in SEQ ID NO.2, Inpp5e cko dsgRNA1 is shown in SEQ ID NO.3, and Inpp5e cko dsgRNA2 is shown in SEQ ID NO.4; (2) Construction of the targeting vector: The targeting vector consists of four fragments: a 1986 bp left homologous arm LHA, an 871 bp middle insert loxp, a 2079 bp right homologous arm RHA, and a 2659 bp plasmid backbone pUC19L. LHA and RHA were obtained by PCR amplification using tail lysate from C57BL / 6J wild-type mice as templates, and loxp was obtained by PCR amplification using the Blunt-inpp5e loxp plasmid as a template. The plasmid backbone was linearized using pUC19L. The four purified and recovered fragments LHA, loxp, RHA, and pUC19L linearized vector were seamlessly spliced in one step using the pEASY®-Basic Seamless Cloning and Assembly Kit to obtain the targeting vector pUC19L-Inpp5e CKO Targeting Vector. Endotoxin-free large-scale extraction was performed using a large-scale plasmid extraction kit. The obtained large-scale plasmid was digested with restriction endonuI and recovered. Kb fragments were used as donors for microinjection; (3) Inpp5e cko usgRNA1, Inpp5e cko dsgRNA1 and Donor were microinjected into mouse zygotes to obtain Inpp5e gene conditional knockout mice.
2. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 1, characterized in that: In step (1), the concentrations of Inpp5e cko usgRNA1, Inpp5e cko usgRNA2, Inpp5e cko dsgRNA1, and Inpp5ecko dsgRNA2 are 3269 ng / μL, 3104 ng / μL, 3069 ng / μL, and 3200 ng / μL, respectively.
3. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 2, characterized in that: The step (2) also includes identification of the target vector bacterial culture: the Blunt-Inpp5e loxp plasmid is constructed by amplifying the Loxp fragment by high-fidelity polymerase PCR, which contains two Loxp fragments and exon7 and exon8. After purification and recovery, it is ligated into the Blunt blunt-end vector; the Blunt-Inpp5e loxp bacterial culture is identified by gel electrophoresis. The bacterial culture of pUC19L-Inpp5e CKO Targeting Vector was identified using three pairs of primers. The product sizes were 740 bp Inpp5e-JDF1+Inpp5e-JDR1, 736 bp Inpp5e-JDF2+Inpp5e-JDR2, and 713 bp Inpp5e-JDF3+Inpp5e-JDR3. The bacterial culture was identified by agarose gel electrophoresis.
4. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 3, characterized in that: Step (2) also includes targeting vector sequencing identification: sequencing the bacterial culture and sequencing the 4936 bp LHA-Loxp-RHA fragment.
5. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 4, characterized in that: In step (3), embryo transfer is performed on the same day as microinjection, and mice are born 21 days after the transfer.
6. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 5, characterized in that: The method also includes step (4), where mice are identified by tail clipping about 10 days after birth; and one Founder is obtained by PCR and sequencing.
7. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 6, characterized in that: In step (4), the identification primer sequences are as follows: Inpp5eCKOcushaiF as shown in SEQ ID NO.5, Inpp5eCKOcushaiR as shown in SEQ ID NO.6, Inpp5eckoCF as shown in SEQ ID NO.7, Inpp5eckoCR as shown in SEQ ID NO.8, Inpp5ecko-RIR3 as shown in SEQ ID NO.9, Inpp5ecko-RIF3 as shown in SEQ ID NO.10, Inpp5e ckouF as shown in SEQ ID NO.11, Inpp5e cko uR as shown in SEQ ID NO.12, Inpp5e cko dF as shown in SEQ ID NO.13, and Inpp5e cko dR as shown in SEQ ID NO.
14.
8. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 7, characterized in that: In step (4), in PCR, Inpp5eCKOcushaiF+Inpp5eCKOcushaiR, Inpp5e-cko-RI-F3+Inpp5e-cko-RI-R3 are performed using a three-step method with Kangwei Century mix; Inpp5eckoCF+Inpp5eckocushaiR, Inpp5eckocushaiF+Inpp5eckoCR, Inpp5eckoCF+Inpp5eckoCR are performed using a two-step method with TOYOBO KODOne PCR Master Mix-Blue.
9. The method for constructing an Inpp5e gene conditional knockout animal model according to claim 8, characterized in that: The method also includes crossbreeding. After the positive Founder mice reached sexual maturity, they were mated with C57BL / 6J mice in the same cage. Four F1 generation positive mice were obtained and identified as 8319♀, 8645♂, 9097♀, and 9100♀. Inpp5e CKO CF+Inpp5eCKO CR was sequenced using Inpp5e cko uF and Inpp5e cko dR. The large fragment sequencing results of 8319♀ showed that there were two peaks at the two Loxp positions, indicating that there were two Loxp fragments. 8319♀ did not contain random insertions. It was amplified using primers Inpp5ecko uF+Inpp5e cko dR. The 1475 bp amplified fragment was ligated into the Blunt vector and positive clones were selected for sequencing. The sequencing results showed that it contained two Loxp sequences of the target sequence. Sequencing results for fragment 8645♂ showed overlapping peaks at two Loxp positions, and Loxp sequences were also present in both the LA and RA fragments. F1 mice 9097♀ and 9100♀ did not contain random insertions. Amplification was performed using primers Inpp5e cko uF+ Inpp5e cko dR, and the 1475 bp amplified fragment was ligated into the Blunt vector. Positive clones were selected for sequencing, and the sequencing results showed that they contained the target sequence at two Loxp positions. Therefore, sequencing results indicate that 8319♀, 8645♂, 9097♀, and 9100♀ are all positive F1 generation mice.
10. The application of Inpp5e gene knockout animal models not for disease diagnosis and treatment purposes, characterized in that, The application includes using the Inpp5e gene knockout animal model obtained by the construction method according to any one of claims 1 to 9 to study neurodevelopmental related diseases.