Construction method and application of Drd3-P2A-CreERT2 gene knock-in mouse model

Abstract

The present invention discloses a method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model and its application, wherein the construction method is: using the CRISPR / Cas9 system to insert a P2A-CreERT2 element before the translation termination site of the Drd3-201 transcript, and CreERT2 is expressed under the regulation of the endogenous Drd3 gene. The present invention uses CRISPR / Cas technology to insert the Cre-ERT fusion protein into the Drd3 gene sequence, realizes the manipulation of the expression of the Drd3 gene in time windows such as the embryonic stage, the developmental stage, and the mature stage, and provides an important tool for the study of the function of the Drd3 gene and the drug screening of related nervous system diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model and an application thereof. Background Art

[0002] Dopamine (DA) is an important neurotransmitter in the central nervous system and is involved in processes such as movement, emotion, and drug addiction. Dopamine functions through five different dopamine receptors, which are divided into D1-like and D2-like receptors according to their pharmacological and structural characteristics. The D1-like receptor family includes dopamine type 1 receptors and dopamine type 5 receptors, which can activate excitatory G protein Gα / s signals and promote the formation of cyclic AMP through adenylate cyclase. The D2-like receptor family includes dopamine type 2, 3, and 4 receptors, which can activate inhibitory G protein Gαi / o signals and inhibit adenylate cyclase from producing cyclic AMP. Dopamine type 3 receptor (Drd3) is mainly expressed in brain regions such as the Calleja island, nucleus accumbens, and olfactory tubercle, and is involved in physiological processes such as motivation, reward, and cognition. Drd3 can be expressed in synaptic structures to regulate metabolic processes such as dopamine synthesis, release, and transmission. Studies have shown that Drd3 is involved in the occurrence and development of various neurological diseases such as depression, Parkinson's disease, schizophrenia, and autism.

[0003] At present, there are few studies on the function of Drd3, and its specific role in diseases such as depression, Parkinson's disease, and schizophrenia is still unknown. Therefore, it is necessary to develop a Drd3 gene knock-in mouse model, which is of great significance for exploring the role of Drd3 in neurological diseases, related mechanisms, and possible molecular pathways involved. It also provides an animal model for drug screening with Drd3 as a molecular target. Drd3 whole-body knockout mice have been widely used to study the physiological functions of Drd3 molecules and their pathological effects in neurological diseases, providing an important research tool for revealing the function of Drd3 protein. However, the development of conditional expression mice Cre mice that mark neuronal groups expressing Drd3 at specific times and specific spaces has not been reported.

[0004] One of the most effective methods for constructing Cre mice is through the Cre-LoxP recombinase system. Specific Cre tool mice can express tissue-specific Cre recombinase under the regulation of a tissue-specific gene promoter. Cre recombinase recognizes the inverted repeat sequences at both ends of the loxP site and combines to form a dimer, and then combines with the dimer on another loxP site to form a tetramer. Since the LoxP site is directional, the DNA sequence between the loxP sites is cut by the Cre recombinase, so the result of the recombination depends on the position and direction of the loxP site. At present, there are four main Cre mouse construction strategies, including plasmid expression vector Cre mouse model, site-directed knock-in Cre mouse model, BAC transgenic Cre mouse model, and safe site knock-in Cre mouse model. Traditionally, the use of cell / tissue-specific promoter partial DNA fragment strategy and random gene insertion results in inaccurate or leaky Cre expression, making it difficult to truly reflect the desired specificity, resulting in off-target cleavage of Cre expression. Summary of the invention

[0005] In view of this, the present invention aims to provide a method for constructing a Drd3 gene knock-in mouse model, so as to manipulate the expression of the Drd3 gene in time windows such as the embryonic period, developmental period, and mature period, and to provide a mouse model for studying the physiological function of Drd3 and its role in depression, anxiety, Alzheimer's disease, Parkinson's disease and other diseases.

[0006] The technical solution of the present invention is as follows:

[0007] A method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model, specifically comprising: using the CRISPR / Cas9 system to insert a P2A-CreERT2 element before the translation termination site of the Drd3-201 transcript, wherein the nucleotide sequence of the P2A-CreERT2 element is shown in SEQ ID NO.1.

[0008] The Drd3 gene (gene ID 13490) is located on chromosome 16, and there are two transcripts Drd3-201 and Drd3-202, wherein the Drd3-201 transcript contains a complete conserved protein-coding region (CCDS); the present invention selects the Drd3-201 transcript design, the transcript encodes 7 exons, the translation start site ATG is located at exon1, and the translation termination site TGA is located at exon7 (its nucleotide sequence is shown in SEQ ID NO.2), the transcript is 1455bps long, and encodes 446 amino acids. The present invention inserts the P2A-CreERT2 element before the translation termination site of the Drd3-201 transcript, and CreERT2 expresses Cre-ERT (a Cre fusion protein with a mutant ligand binding domain of an estrogen receptor) under the regulation of the endogenous Drd3 gene. The present invention can achieve temporal control of Cre activity through Cre-ERT, and the protein can be activated by tamoxifen induction, making in vivo gene editing more spatiotemporally flexible. At the same time, the present invention adopts the P2A peptide connection knock-in method. Without performing a new translation program, the translation of the cre recombinase can be synchronized with the translation of the original vector gene. After the completion, P2A automatically breaks to separate the cre recombinase from the target gene, thereby achieving the goal of not destroying the expression of the Drd3 gene while not interfering with the expression level of Cre.

[0009] Preferably, the above construction method includes the following steps:

[0010] Design, construct and in vitro transcribe gRNA, and construct homologous recombination vector (Donor vector);

[0011] The CRISPR / Cas9 system and Donor vector samples were introduced into mouse fertilized eggs, which were then transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth. The recipient mice gave birth to the F0 generation, and the genotypes were confirmed. After the positive F0 generation mice reached sexual maturity, they were mated with wild-type background mice to give birth to the F1 generation, and the genotypes were confirmed.

[0012] More preferably, in the above construction method, the gRNA sequence is as shown in SEQ ID NO.3-4.

[0013] More preferably, in the above construction method, the CRISPR / Cas9 system and homologous recombination vector sample are introduced into mouse fertilized eggs by microinjection.

[0014] More preferably, in the above construction method, the CRISPR / Cas9 system is a T7-Cas9-gRNA expression vector, which can transcribe gRNA and Cas9 mRNA in vitro under the action of T7 RNA polymerase.

[0015] More preferably, in the above construction method, the primers used to confirm the genotype include F1 / R1, F2 / R2 and F3 / R3, wherein the sequence of F1 / R1 is shown in SEQ ID NO.3-4, the sequence of F2 / R2 is shown in SEQ ID NO.5-6, and the sequence of F3 / R3 is shown in SEQ ID NO.7-8. Based on the above primer pairs, the method for determining the genotype is as follows: for the wild type, no Target band can be obtained when PCR reactions are performed with F1 / R1 and F2 / R2, respectively, and a single WT band is obtained when PCR reactions are performed with F3 / R3; for the heterozygote, the Target band is obtained when PCR reactions are performed with F1 / R1 and F2 / R2, respectively; and a single WT band is obtained when PCR reactions are performed with F3 / R3; for the homozygote, the Target band is obtained when PCR reactions are performed with F1 / R1 and F2 / R2, respectively; and a single WT band is not obtained when PCR reactions are performed with F3 / R3; wherein, the length of the Target band corresponding to F1 / R1 is 2166 bp, the length of the Target band corresponding to F2 / R2 is 2078 bp, and the length of the WT band corresponding to F3 / R3 is 294 bp.

[0016] More preferably, in the above construction method, the mouse fertilized egg has a C57BL / 6JGpt background.

[0017] The Drd3-P2A-CreERT2 gene knock-in mouse model constructed based on the method of the present invention can be used to study the function of Drd3 and its mechanism of action in depression, anxiety, Alzheimer's disease, Parkinson's disease and other diseases, and can also be used to screen drugs with Drd3 as a molecular target.

[0018] The beneficial effects of the present invention are:

[0019] The present invention utilizes CRISPR / Cas technology to site-specifically knock-in a single copy of Cre into the Drd3 gene sequence, which can not only increase the accuracy of Cre gene expression activity and effectively reduce the off-target activity of the Cre enzyme, but also manipulate the expression of the Drd3 gene in a specific time and space. For the first time, a conditional expression mouse, the Cre mouse, which can mark a neuronal group expressing Drd3 at a specific time and space, was successfully constructed, providing an important tool for the functional research of Drd3 and the development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the strategy for constructing the Drd3-P2A-CreERT2 gene knock-in mouse model of the present invention.

[0021] Figure 2 Schematic diagram of the strategy for identifying genotypes by PCR in Example 1.

[0022] Figure 3 This is a partial electrophoresis result diagram of F1 generation mice in Example 1; in the figure, N is a blank control, M is a DNA marker, and 32, 33, 35 and 37 are heterozygous mice. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0025] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0026] Example 1

[0027] This example provides a method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model based on the CRISPR / Cas9 system. Figure 1 As shown, specifically: using the CRISPR / Cas9 system, the P2A-CreERT2 element is inserted before the translation termination site TGA of the Drd3-201 transcript, and CreERT2 is expressed under the regulation of the endogenous Drd3 gene. The method specifically includes the following steps:

[0028] The mouse Drd3 gene sequence was obtained from the NCBI website, and the transcript Drd3-201 (ENSMUST00000023390.5) sequence was selected as the target sequence; a pair of 20 bp oligonucleotide chain sequences for the target DNA were designed according to the score to prepare gRNA, and the designed sequence was synthesized into the product of PAGE. The gRNA sequences used in this example are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0029] A donor vector carrying the target site homology region and the target fragment is designed according to the gRNA sequence, and primers are designed in the target region for subsequent gene identification of mice.

[0030] The two synthesized single-stranded oligonucleotide gRNA sequences were annealed to form double-stranded DNA, and the gRNA expression vector was constructed. The recombinant plasmid was transformed into DH5a competent cells. The positive clone plasmid was screened and identified by kanamycin resistance and sequencing of target DNA, and the correct colony clone was selected. After expansion culture, the plasmid was extracted for preparing in vitro transcription template; a recombinant plasmid carrying the homologous region of the target site and the target fragment (Donor vector, which contains the sequence shown in SEQ ID NO.11) was prepared, and the recombinant plasmid was transformed into DH5a competent cells. The positive clone plasmid was screened and identified by ampicillin resistance and sequencing of the inserted fragment, and the correct colony clone was selected. After expansion culture, the plasmid was extracted and purified, and the obtained donor fragment product was used for injection.

[0031] A T7-Cas9-gRNA expression vector was constructed, which can transcribe gRNA and Cas9mRNA under the action of T7 RNA polymerase for microinjection of fertilized eggs and embryos.

[0032] Six-week-old C57BL / 6 mice were selected as donor female mice. After superovulation, they were caged with 10-week-old C57BL / 6 male mice. The donor C57BL / 6 female mice with vaginal plugs were used to collect fertilized eggs and used as embryo transplant donors. The T7-Cas9-gRNA expression vector and the Donor vector sample were microinjected into the fertilized eggs of mice with C57BL / 6JGpt background; 10-20 fertilized eggs with good morphology that survived the injection were selected and cultured into embryos, and then transplanted into pseudo-pregnant ICR female mice with vaginal plugs, waiting for them to become pregnant and give birth.

[0033] The F0 generation pups born by the recipient mice were numbered after their tails and toes were cut off at 5-7 days, and their genomic DNA was extracted for PCR and sequencing to confirm their genotype. After the positive F0 generation mice reached sexual maturity, they were mated with wild-type background mice; the F1 generation mice born were numbered after their tails and toes were cut off at 5-7 days, and their genomic DNA was extracted for PCR and sequencing to confirm their genotype.

[0034] PCR identification strategy Figure 2As shown, the primers used are shown in Table 1 (in the figure, F1 is primer T064748-Drd3-5tF1, R1 is primer CreERT2-5tR1, F2 is CreERT2-3tF1, R2 is T064748-Drd3-3tR1, F3 is T064748-Drd3-WT-tF1, and R3 is T064748-Drd3-WT-tR1). The genotype result judgment method is: for wild type, ① and ② PCR reactions did not obtain the Target band, and ③ PCR reaction could obtain a single WT band; for heterozygotes, ① and ② PCR reactions could obtain the Target band, and ③ PCR reaction could obtain a single WT band; for homozygotes, ① and ② PCR reactions could obtain the Target band, and ③ PCR reaction did not obtain a single WT band.

[0035] Table 1 Primer sequence information

[0036]

[0037] Specifically, the PCR reaction system and reaction procedure used for genotype detection are shown in Table 3 and Table 4, respectively.

[0038] Table 3 PCR reaction system

[0039]

[0040] Table 4 PCR reaction procedure

[0041]

[0042] The above method was used to identify the genotype of F1 mice. Some electrophoresis results are shown in the figure below. Figure 3 As shown: F1 mice numbered 32, 33, 35 and 37 are heterozygous mice, and are consistent with the sequencing results.

[0043] In summary, the method of the present invention successfully established a Drd3 gene knock-in mouse model, which is of great significance for studying the physiological function of Drd3 and also provides an animal model for drug screening with Drd3 as a molecular target.

[0044] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model, characterized in that: The P2A-CreERT2 element is inserted before the translation termination site of the Drd3-201 transcript using the CRISPR / Cas9 system, wherein the nucleotide sequence of the P2A-CreERT2 element is shown in SEQ ID NO.1; the construction method comprises the following steps: Design, construct and in vitro transcribe gRNA, and construct homologous recombination vector; The CRISPR / Cas9 system and homologous recombination vector samples are introduced into mouse fertilized eggs, which are then transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth. The recipient mice give birth to the F0 generation, and their genotypes are confirmed. After the positive F0 generation mice reach sexual maturity, they are mated with wild-type background mice to give birth to the F1 generation, and their genotypes are confirmed. The resulting heterozygotes are the mouse model. Among them, the sequence of the Drd3-201 transcript is shown in ENSMUST00000023390.5, the gRNA sequence is shown in SEQ ID NO.3-4, and the CRISPR / Cas9 system includes gRNA and Cas9 mRNA.

2. The construction method according to claim 1, characterized in that: The CRISPR / Cas9 system and homologous recombination vector samples were introduced into mouse fertilized eggs by microinjection.

3. The construction method according to claim 1, characterized in that: The primers used to confirm the genotype include F1 / R1, F2 / R2 and F3 / R3, wherein the sequence of F1 / R1 is shown in SEQ ID NO.5-6, the sequence of F2 / R2 is shown in SEQ ID NO.7-8, and the sequence of F3 / R3 is shown in SEQ ID NO.9-10.

4. The construction method according to claim 3, characterized in that: The method for determining the genotype is: For the wild type, no Target band was obtained when PCR reactions were performed with F1 / R1 and F2 / R2, and a single WT band was obtained when PCR reactions were performed with F3 / R3; For heterozygotes, the Target band was obtained when PCR reactions were performed with F1 / R1 and F2 / R2, respectively; a single WT band was obtained when PCR reactions were performed with F3 / R3; Homozygotes, Target bands were obtained when PCR reactions were performed with F1 / R1 and F2 / R2, respectively; no single WT band was obtained when PCR reactions were performed with F3 / R3; Among them, the length of the Target band corresponding to F1 / R1 is 2166bp, the length of the Target band corresponding to F2 / R2 is 2078bp, and the length of the WT band corresponding to F3 / R3 is 294bp.

5. The construction method according to claim 1, characterized in that: The mouse fertilized eggs had a C57BL / 6JGpt background.

6. Use of the Drd3-P2A-CreERT2 gene knock-in mouse model prepared according to the construction method according to any one of claims 1 to 5 in screening drugs with Drd3 as a molecular target.

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