A Kdf1 gene conditional knockout mouse model and its construction method

Abstract

The present invention relates to the technical field of genetically engineered mouse models, and specifically to a Kdf1 gene conditional knockout mouse model and a construction method thereof. The construction method comprises the following steps performed in sequence: obtaining a donor vector; obtaining gRNA; obtaining a conditional knockout mouse model capable of accurately knocking out the Kdf1 gene. The present technical solution solves the technical problem that it is difficult to accurately study the mechanism of Kdf1 regulating organ development using the Kdf1 gene functional defect mouse model of the prior art. The mouse model of the present solution can be mated and propagated with Cre mice from a variety of tissue sources, thereby constructing a mouse model of Kdf1 gene knockout with multiple tissue specificities that can be stably propagated, thereby avoiding embryonic lethality caused by systemic organ dysfunction in mice, and at the same time, the Kdf1 gene of the tooth germ epithelial tissue can be specifically knocked out, creating conditions for studying the function of the Kdf1 gene.

Description

Technical Field

[0001] The present invention relates to the technical field of genetically engineered mouse models, and in particular to a Kdf1 gene conditional knockout mouse model and a construction method thereof. Background Art

[0002] The human Kdf1 gene (keratinocyte differentiation factor 1) was discovered in 2013 and is located on human chromosome 1p36.11. It can encode a protein with 398 amino acids. The mouse Kdf1 protein has 90% homology with the human Kdf1 (C1orf172) protein, which lays a very favorable foundation for using mouse models to study the function of the human KDF1 gene. Although the role of the Kdf1 gene in mouse skin development has been preliminarily determined, and it has been found that the Kdf1 protein is specifically expressed in the tooth germ epithelium and is almost not expressed in the mesenchyme, the developmental role of the Kdf1 gene in parts such as teeth and oral mucosal epithelium that are homologous to skin development is still unclear. So far, there are only three reports on Kdf1 gene mutations and human congenital tooth agenesis. The function of the Kdf1 gene and its pathogenicity urgently need further study.

[0003] Constructing a mouse model with Kdf1 gene deficiency is an important means to study the function of Kdf1 gene and its pathogenicity. In 2013, Lee et al. first reported a mouse model with systemic Kdf1 functional deficiency—shd mouse. The technical means were: using N-ethyl-N-nitrosourea (ENU) to systemically induce T>G base transversion in the second intron of Kdf1 gene, resulting in a change in the reading frame of Kdf1 protein isoform 1 and a frameshift mutation with an extended amino acid sequence, or an in-frame deletion mutation of Kdf1 protein isoform 2 and a truncation mutation, thereby affecting the function of mouse Kdf1 gene. Subsequently, the researchers also used gene trapping technology to knock down Kdf1 (1810019J16Rik tm1a(EUCOMM)Wtsi , Kdf1 GT ) were introduced into mouse embryonic stem cells to establish Kdf1 gene base substitution mutation GT / GTThe systemic phenotype of this mouse model is highly consistent with that of shd mice, which further confirms that the developmental defects of both mice are caused by the loss of Kdf1 gene function. However, the mouse model constructed in the above study is a systemic Kdf1 gene function defect, which means that the Kdf1 gene expressed in different developmental tissues of the mouse body loses its function. Therefore, the systemic tissue and organ disorders of this mouse model lead to perinatal death. This mouse model lacks the tissue specificity of gene knockout, and it is difficult to accurately study the role and mechanism of Kdf1 gene in the development of different tissues and organs. In addition, using existing technologies, such as gene mutation mouse models established by drug induction such as ENU or gene mutation mouse models established based on mouse embryonic stem cells through trapping vectors, there are problems such as difficult prediction of gene mutation points, inability to specifically knock out specific source cells, low knockout rate, and complex knockout methods. It is urgent to construct a new Kdf1 gene defect mouse model to meet the needs of accurate research on the mechanism of Kdf1 gene regulating tooth germ and oral epithelial development. Summary of the invention

[0004] The present invention aims to provide a method for constructing a Kdf1 gene conditional knockout mouse model to solve the technical problem that it is difficult to accurately study the mechanism of Kdf1 regulating tooth germ and oral epithelial development using the Kdf1 gene defective mouse model in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for constructing a Kdf1 gene conditional knockout mouse model comprises the following steps performed in sequence:

[0007] S1: Obtaining a donor vector: The donor vector includes an upstream loxP site and a downstream loxP site; exon 2 and exon 4 of the Kdf1 gene are located between the upstream loxP site and the downstream loxP site;

[0008] S2: Obtaining gRNA;

[0009] S3: Obtain Kdf1 gene conditional knockout mouse model.

[0010] This protocol also provides a method for constructing a Kdf1 gene conditional knockout mouse model, wherein the genotype is Kdf1 flox / flox Kdf1 conditional knockout mouse model.

[0011] The principles and advantages of this solution are:

[0012] This technical solution uses crisper-Cas9 technology to construct a conditional knockout mouse model of the Kdf1 gene. By constructing a donor vector containing two loxP sites and then combining crisper-Cas9 technology to transform the mouse fertilized egg genome, F0 generation mice containing the conditionally knockout target gene are obtained. After multiple generations of hybridization and breeding, a stable Kdf1 gene can be obtained. flox / flox When using tissue-specific Cre, such as K14-Cre mice or Pitx2-Cre mice with Kdf1 flox / flox Mating mice can completely inactivate their gene functions, achieving a stable and efficient gene knockout effect. Among them, Cre-loxP recombination is a mature technology in the existing technology. It is a site-specific recombinase technology that can perform deletion, insertion, translocation and inversion at specific sites of DNA. This system can be used to modify DNA in cells for specific cell types or using specific external stimuli.

[0013] In summary, the beneficial effects of this technical solution are:

[0014] (1) Kdf1 of the present invention flox / flox Mouse models can be mated with Cre recombinase mice from various tissue sources to construct mice with multiple tissue-specific knockouts of the Kdf1 gene, thus avoiding embryonic lethality caused by systemic organ dysfunction in mice. On the other hand, the Kdf1 gene is only expressed in epithelial tissues such as tooth germ epithelium. Specific knockout of the Kdf1 gene in tooth germ epithelium is conducive to the precise study of the mechanism of the Kdf1 gene regulating the development of tooth germs and oral epithelium.

[0015] (2) The crisper-Cas9 technology is very simple, convenient and low-cost in construction and use. There is no integration of exogenous DNA in the entire targeting process, avoiding the biosafety issues caused by traditional transgenics. On the basis of accurately knocking out genes in the animal genome, the expression and interaction of different genes and molecules can be clarified, so as to deeply understand the function and regulatory network of the studied genes.

[0016] (3) The Kdf1 gene has four exons (exon 1-4) and five transcripts. The inventors' previous studies found that the entire coding sequence of the Kdf1 gene can be knocked out by using the second to fourth exons of the Kdf1 gene as target fragments, making its function completely inactivated. When designing loxP sites, the insertion of loxP sites should avoid affecting the expression of the target gene (before mating with Cre tool mice), and considering the complete inactivation of the Kdf1 gene after mating with Cre tool mice, this scheme chooses to insert the upstream loxP at a non-conserved site upstream of exon 2 and the downstream loxP at a non-conserved site downstream of exon 4, thereby avoiding the influence of loxP insertion on the expression of the target gene.

[0017] Further, in S1, the donor vector includes an upstream loxP site located 839-873 bp upstream of exon 2 of the Kdf1 gene and a downstream loxP site located 560-594 bp downstream of exon 4 of the Kdf1 gene.

[0018] When designing loxP sites, the insertion of loxP sites should avoid affecting the expression of the target gene (before mating with Cre tool mice), and considering the complete inactivation of the Kdf1 gene after mating with Cre tool mice, this solution chooses to insert the upstream loxP at the non-conservative site at 839-873bp upstream of exon 2, and insert the downstream loxP at the non-conservative site at 560-594bp downstream of exon 4, so as to avoid the influence of the insertion of loxP on the expression of the target gene. In this technical solution, knocking out exon2-4 is the best choice, which can affect all transcripts (all five transcripts are affected). In addition, only by inserting loxp into these two specific non-conservative sites can the complete knockout of exon2-4 be achieved, and the function of the gene before the conditional knockout will not be affected, and the correct shearing and mouse survival rate will not be affected.

[0019] Further, in S1, the donor vector includes a 5' homology arm region as shown in SEQ ID NO.2, an upstream loxP site as shown in SEQ ID NO.4, a selective knockout as shown in SEQ ID NO.1, a downstream loxP site as shown in SEQ ID NO.4, and a 3' homology arm region as shown in SEQ ID NO.3, which are arranged in sequence from the 5' end to the 3' end.

[0020] By adopting the above technical solution, the design of the donor vector can also avoid the arm and cKO containing sequences that completely match the gRNA, so as to avoid the off-target problem that the gRNA may cause to the donor vector or homologous recombination repair site. The above design also avoids the insertion of loxP sites and should avoid affecting the expression of the target gene.

[0021] Further, in S2, the gRNA includes gRNA1 whose gene sequence is shown in SEQ ID NO.5, gRNA2 whose gene sequence is shown in SEQ ID NO.6, gRNA3 whose gene sequence is shown in SEQ ID NO.7, and gRNA4 whose gene sequence is shown in SEQ ID NO.8.

[0022] Further, in S2, the gRNA consists of gRNA1 with a gene sequence as shown in SEQ ID NO. 5 and gRNA2 with a gene sequence as shown in SEQ ID NO. 6. After off-target analysis, gRNA1 and gRNA2 are the optimal gRNAs.

[0023] Furthermore, the S3 includes a microinjection step, a positive F0 mouse acquisition step, an F1 generation mouse acquisition step, and an F2 generation mouse acquisition step; in the microinjection step, the Donor vector, gRNA, and Cas9 protein are co-injected into the mouse fertilized egg; the microinjected fertilized egg is returned to the surrogate mouse oviduct; after the mouse is born, it is identified and screened to obtain a positive F0 mouse. The exogenous gene is transferred into the fertilized egg by microinjection to obtain a transgenic mouse.

[0024] Furthermore, in the step of obtaining positive F0 mice, the fertilized eggs after microinjection were returned to the oviduct of the surrogate mouse; after the mice were born, they were screened by PCR and sequencing to obtain positive F0 mice. During the microinjection process, a total of 550 fertilized eggs were injected and returned to the oviduct of the surrogate mother mouse, and a total of 64 F0 mice were born, with a birth rate of 12%, 5 fixed-point positive F0 mice, a positive rate of 7.8%, and a survival rate of 100%.

[0025] Furthermore, in the step of obtaining F1 generation mice, sexually mature positive F0 mice were bred with wild-type mice for one generation, and the genotype of Kdf1 was screened by PCR. flox / + F1 generation mice. Genetically stable transgenic mouse offspring were obtained through hybridization.

[0026] Furthermore, in the step of obtaining F2 mice, the genotype of Kdf1 flox / + The F1 generation mice were inbred and the genotype of Kdf1 was screened by PCR. flox / flox F2 generation mice. Homozygous F2 generation mice were obtained through inbreeding and can be used for subsequent conditional gene removal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the sequence of the donor vector of Example 1 of the present invention (part 1).

[0028] Figure 2 This is the sequence of the donor vector of Example 1 of the present invention (part 2).

[0029] Figure 3 This is the sequence of the donor vector of Example 1 of the present invention (part 3).

[0030] Figure 4 This is the sequence of the donor vector of Example 1 of the present invention (Part 4).

[0031] Figure 5 This is the sequence of the donor vector of Example 1 of the present invention (Part 5).

[0032] Figure 6 It is a schematic diagram of the structure of the donor vector of Example 1 of the present invention, the wild-type allele of the Kdf1 gene, the mutant allele containing cKO, and the mutant allele after Cre recombination.

[0033] Figure 7 This is the chromosome localization analysis of gRNA1 in Example 1 of the present invention.

[0034] Figure 8 This is the chromosome localization analysis of gRNA2 in Example 1 of the present invention.

[0035] Fig. 9 This is the chromosome localization analysis of gRNA3 in Example 1 of the present invention.

[0036] Fig.10 This is the chromosome localization analysis of gRNA4 in Example 1 of the present invention.

[0037] Fig.11 This is the off-target analysis of gRNA1 in Example 1 of the present invention.

[0038] Fig.12 This is the off-target analysis of gRNA2 in Example 1 of the present invention.

[0039] Fig.13 This is the off-target analysis of gRNA3 in Example 1 of the present invention.

[0040] Fig.14 This is the off-target analysis of gRNA4 in Example 1 of the present invention.

[0041] Fig.15 Schematic diagram of the RCR and sequencing primer binding sites of Example 2 of the present invention (F1 generation).

[0042] Fig.16 This is the electrophoresis diagram of the PCR verification of Example 2 of the present invention (PCR primer pair 1).

[0043] Fig.17This is the electrophoresis diagram of the PCR verification of Example 2 of the present invention (PCR primer pair 2).

[0044] Fig.18 This is a diagram of the sequencing results of Example 2 of the present invention (F1 generation).

[0045] Fig.19 Schematic diagram of the RCR primer binding site of Example 2 of the present invention (first identification of F0 generation).

[0046] Fig. 20 This is the first identification electrophoresis diagram of Example 2 of the present invention (primer F5R4).

[0047] Fig.21 This is the first identification electrophoresis diagram of Example 2 of the present invention (primer F2R3).

[0048] Fig. 22 This is the second identification electrophoresis diagram of Example 2 of the present invention (primer F1R1).

[0049] Fig.23 This is the second identification electrophoresis diagram of Example 2 of the present invention (primer F2R2).

[0050] Fig.24 This is the electrophoresis diagram for F2 generation identification in Example 2 of the present invention. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with the embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art: the materials and reagents used can all be obtained from commercial channels. In the experimental example, the mouse genome was extracted using the TaKaRa kit (TaKaRaMiniBEST Universal Genomic DNA Extraction kit), and the mouse tail was taken for genome extraction.

[0052] Example 1: Vector and gRNA (guide-RNA) design and construction

[0053] The receiving number of Kdf1 gene is NM_001083916.1, Kdf1 flox / flox The mouse model construction process includes the following steps:

[0054] This step mainly includes designing the gRNA sequence and targeting vector scheme; using In-Fusion technology to construct a donor vector (targeting vector) including loxP sites, and verifying the targeting vector through enzyme digestion, PCR and sequencing.

[0055] The sequence of the donor vector is shown in Figure 1-Figure 5The donor vector includes a 5' homology arm region (5'arm), an upstream loxP site, a selective knockout (cKO), a downstream loxP site, and a 3' homology arm region (3'arm). Figure 1-Figure 5 The light-colored part in the figure is the cKO region (SEQ ID NO.1), and the single underlined part in the light-colored region is the exon (exon 2-4 in order from front to back); the dark-colored wavy line marked region is the 5'arm (SEQ ID NO.2) and 3'arm (SEQ ID NO.3), and the double underlined region is the two loxP sites (SEQ ID NO.4). The Kdf1 gene has four exons (exon1-4) and five transcripts. The inventors found in their previous studies that the entire coding sequence of the Kdf1 gene can be knocked out by using the second exon to the fourth exon of the Kdf1 gene as the target fragment, making its function completely inactivated. When designing loxP sites, the insertion of loxP sites should avoid affecting the expression of the target gene (before mating with Cre tool mice), and considering the complete inactivation of the Kdf1 gene after mating with Cre tool mice, this scheme chooses to insert the upstream loxP at the non-conservative site 839-873bp upstream of exon 2 and the downstream loxP at the non-conservative site 560-594bp downstream of exon 4, so as to avoid the influence of loxP insertion on the expression of the target gene (see Figure 6 as well as Figure 1-5). In the present technical scheme, knocking out exon2-4 is the best choice. If only exon 2 or exon 2 and 3 are knocked out, some gene coding regions are still left, and the comprehensive inactivation of Kdf1 gene function cannot be achieved. In addition, only by inserting loxp into these two specific non-conservative sites can the complete knockout of exon2-4 be achieved, and the function of the gene before conditional knockout will not be affected, and the correct shearing and mouse survival rate will not be affected. The search for loxp insertion sites also needs to take into account the gRNA off-target score, that is, the confirmation of loxp insertion sites needs to be considered according to the specific circumstances of gRNA design. The design of the above-mentioned donor vector can also avoid arm and cKO containing sequences that fully match the same gRNA, so as to avoid the off-target problem that gRNA may produce to the donor vector or homologous recombination repair site. The inventor has tried other loxp insertion sites, all of which cannot meet the above requirements, and will bring about the experimental results that the birth rate, positive rate and survival rate of the fixed-point positive F0 mice are far lower than those of Example 2, and the growth state of the fixed-point positive F0 mice is not ideal. Only the non-conserved sites at 839-873 bp upstream of exon 2 and 560-594 bp downstream of exon 4 can meet the requirements of this technical solution. Therefore, the confirmation of loxP insertion sites is the result of a large number of experimental studies, and it is difficult to evaluate the effect of loxP insertion site selection before actually conducting experiments.

[0056] gRNA target sequence:

[0057] gRNA1 (paired with the antisense strand of the gene): 5'-GGACGCCTCGTCTGCTCATA GGG -3', (SEQ ID NO.5, located at chr4:133527089-133527111, Figure 7 , reference database: Mus musculus GRCm38 / mm10);

[0058] gRNA2 (paired with the positive strand of the gene): 5'-CTGCTGTATTATCCGGGTCT AGG -3', (SEQ ID NO.6, located at chr4:133531335-133531357, Figure 8 ).

[0059] In the process of selecting gRNA target sequences, the inventors tried a variety of target sequences, including the following two sequences:

[0060] gRNA3 (paired with the positive strand of the gene): 5'-GAGTGCCCTATGAGCAGACG AGG-3', (SEQ ID NO.7, located at chr4:133527084-133527106, Fig. 9 );

[0061] gRNA4 (paired with the positive strand of the gene): 5'-TGGGTGCTGCTGTATTATCC GGG -3', (SEQ ID NO.8, located at chr4:133531329-133531351, Fig.10 ).

[0062] For off-target analysis results of gRNA1-4, see Figure 11-Figure 14 , gRNA1 and gRNA2 are the optimal gRNAs.

[0063] Example 2: Microinjection and identification of F0 mice, breeding and screening of F1 generation mice

[0064] This step mainly includes co-injecting the Donor vector, gRNA (gRNA1 and gRNA2) and Cas9 protein (purchased from NewEngland Biolabs, catalog number M0646M) into the fertilized egg by conventional means of the prior art. More specifically, the solution in tube 1 (to obtain a mixture of gRNA and Cas9 protein) is prepared: 0.8uL 100pmol / uL CrRNA (0.4ul+0.4ul) is added to 5.2uL RNase-free water, and then 0.6uL 100pmol / uL TracRNA is added and mixed and incubated for 5min, and then 0.2uL Cas9 protein (NEB, catalog number: M0646M) is added and mixed and incubated for 10min to obtain the solution in tube 1 (to form gRNA1 and gRNA2); the solution in tube 2 is prepared: the Donor plasmid with a final concentration of 15ng / uL; the solution in tube 1 and the solution in tube 2 are mixed in equal volumes to obtain the RNP injection complex, which is injected into the mouse fertilized egg. Among them, crRNA is CRISPR RNA, and tracrRNA (trans-activating crRNA) is trans-activating crRNA; in the CRISPR / Cas9 system, crRNA will combine with tracrRNA to form gRNA (guide RNA), and then the Cas9 protein will accurately locate the target site with the assistance of gRNA, and finally complete the precise editing of the DNA double strand. Both tracrRNA and crRNA can be designed and obtained through conventional means of existing technology after the gRNA target sequence is confirmed, and can be commissioned to relevant biotechnology companies for synthesis through commercial means. The fertilized eggs after microinjection are returned to the oviduct of the surrogate mouse; after the mouse is born, PCR and sequencing are performed to obtain positive F0 mice.

[0065] The sexually mature positive F0 mice were bred with wild-type mice for one generation, and the genotype of Kdf1 was screened by PCR. flox / + The primer sequences for the F1 generation of mice are:

[0066] PCR Primer Pair 1:

[0067] 5′arm forward primer (F1): 5′-TCCTATTGACCTGCCCCTTAGCG-3′;

[0068] 3′loxP reverse primer (R1): 5′-GTGGATTCGGACCAGTCTGA-3′;

[0069] PCR Primer Pair 2:

[0070] 5′loxP forward primer (F2): 5′-GTGCCCTATGAGCAGACATAACT-3′;

[0071] 3'arm reverse primer (R2): 5'-AGACTGCCCTGCCCACTTGC-3'.

[0072] Schematic diagram of the amplification sites of PCR primer pair 1 and PCR primer pair 2. Fig.15 The electrophoresis results after amplification can be found in Fig.16 and Fig.17 After constructing the F1 generation flox heterozygous mice, the primers F1R1 and F2R2 were used for verification. The primer pair can cover the entire region and can identify whether the recombination is correct and complete (there are the following possibilities: completely correct recombination, no recombination, and incomplete recombination). In addition, sequencing was also performed (see F1 generation sequencing results for details). Fig.18 ) and southern verification to further confirm that the target sequence is completely correctly recombined. The above experimental results have verified that the mouse was successfully constructed, but if the subsequent breeding is still completely verified according to this process, the process is very troublesome. Therefore, a simpler verification and identification method is used in the future. Only local verification is needed to confirm whether the target sequence exists (see below for details).

[0073] Sequencing verification was performed at the same time, and the sequencing primers included:

[0074] 5′ sequencing primer (F3): 5′-CTCCTCTGACCTCCGTGGGC-3′;

[0075] 3' sequencing primer (F4): 5'-AGCCTCATGTTTAAAGACTTCCCT-3'.

[0076] Typical sequencing results can be found in Fig.14The sequencing and electrophoresis results showed that the mutant allele containing cKO was successfully constructed.

[0077] During the microinjection process, a total of 550 fertilized eggs were injected and returned to the oviduct of the surrogate mother mouse. A total of 64 F0 mice were born, with a birth rate of 12%, 5 fixed-point positive F0 mice, a positive rate of 7.8%, and a survival rate of 100%. Among them, the F0 mice were identified twice. The schematic diagram of the amplification site for the first identification can be found in Fig.19 , the schematic diagram of the amplification sites for secondary identification can be found in Fig.15 (The primers are the same as those for the previous F1 identification, namely F1R1 and F2R2 primers.) Positive F0 mice can be successfully bred with wild-type mice to obtain offspring with the genotype of Kdf1 flox / + The primer pairs used in the first identification were F2 and R3 (amplifying the target allele 336 bp) and F5 and R4 (see below, amplifying the target allele 216 bp, the wild-type allele 142 bp).

[0078] Reverse primer (R3): 5'-CTGGCTGGCTCAGCAATTAAGAAT-3'.

[0079] F0 primary identification results (primer F5R4) see Fig. 20 ; F0 first identification results (primer F2R3) see Fig.21 (A total of 44 samples were tested, and some of the sample electrophoresis images are selected as examples.) F0 secondary identification results (primer F1R1) are shown in Fig. 22 ; F0 first identification results (primer F2R2) see Fig.23 (Select samples No. 42 / 14 / 21 / 23 / 44 / 2 / 3 / 4).

[0080] The F1 generation mice were further bred (inbreeding) and the genotype of Kdf1 was screened by PCR experiment method. flox / flox The primer sequences for the F2 generation mice are:

[0081] 5′ forward primer (F5): 5′-AGTTTCCTAATGGACACTACAGGAC-3′;

[0082] 3' reverse primer (R4): 5'-CATGCTAGGCAGCACTATGGAC-3'.

[0083] The electrophoresis results showed that Kdf1 flox / flox It is a 216bp electrophoresis band; Kdf1 flox / + There are two bands of 216 bp and 142 bp; Kdf1 + / + It is a 142bp electrophoresis band. Fig.24 .

[0084] When the above scheme uses crisper-Cas9 technology to construct a conditional knockout mouse model of the Kdf1 gene, all coding region exons of the Kdf1 gene in the mouse model will be knocked out, making its gene function completely inactivated, achieving a stable and efficient gene knockout effect. Compared with traditional methods, the construction process is simplified, the cost is low, the cycle is short, and there is no integration of exogenous DNA during the entire targeting process, avoiding the biosafety issues caused by traditional transgenics. When using tissue-specific Cre, such as K14-Cre mice or Pitx2-Cre mice and Kdf1 flox / flox The conditional knockout mice produced by mouse mating have tissue-specific knockout, which is conducive to the precise study of the mechanism of Kdf1 gene regulating tooth germ and oral epithelial development.

[0085] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: The method includes the following steps in sequence: S1: Obtaining a donor vector: The donor vector includes an upstream loxP site and a downstream loxP site; Kdf1 Exons 2 and 4 of the gene are located between the upstream loxP site and the downstream loxP site; The donor vector comprises a 5' homology arm region, a sequence such as SEQ ID NO.2, an upstream loxP site, a selective knockout region, a downstream loxP site, and a 3' homology arm region, which are arranged sequentially from the 5' end to the 3' end; S2: obtaining gRNA; the gRNA is composed of gRNA1 with a gene sequence as shown in SEQ ID NO.5 and gRNA2 with a gene sequence as shown in SEQ ID NO.6; S3: Get Kdf1 Conditional knockout mouse models.

2. A method according to claim 1 Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: In S2, the gRNA also includes gRNA3 whose gene sequence is shown as SEQ ID NO.7 and gRNA4 whose gene sequence is shown as SEQ ID NO.

8.

3. A method according to claim 1 Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: The S3 includes a microinjection step, a positive F0 mouse acquisition step, an F1 generation mouse acquisition step, and an F2 generation mouse acquisition step; in the microinjection step, the Donor vector, gRNA, and Cas9 protein are co-injected into mouse fertilized eggs.

4. A method according to claim 3 Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: In the step of obtaining positive F0 mice, the fertilized eggs after microinjection are returned to the oviduct of the surrogate mice; after the mice are born, they are screened by PCR and sequencing to obtain positive F0 mice.

5. A method according to claim 4 Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: In the step of obtaining F1 generation mice, sexually mature positive F0 mice were bred with wild-type mice for one generation, and the genotype of Kdf1 flox / + F1 generation mice.

6. A method according to claim 5 Kdf1 A method for constructing a gene conditional knockout mouse model, characterized in that: In the step of obtaining F2 generation mice, the genotype Kdf1 flox / + The F1 generation mice were inbred for one generation, and the genotype was screened by PCR. Kdf1 flox / flox F2 generation mice.

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