CN116144709A - Kdf1 gene conditional knockout mouse model and construction method thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering mouse models, in particular to a Kdf1 gene conditional knockout mouse model and a construction method thereof. The construction method comprises the following steps in sequence: obtaining a donor vector; obtaining gRNA; obtaining a conditional knockout mouse model capable of accurately knocking out the Kdf1 gene. The technical scheme solves the technical problem that the Kdf1 gene function defect mouse model in the prior art is difficult to realize accurate research on the mechanism of action of Kdf1 in regulating organ development. The mouse model of the scheme can be subjected to mating passage with Cre mice of various tissue sources, so that a mouse model capable of stably passaging for knocking out Kdf1 genes by various tissue specificities is constructed, embryonic lethality caused by whole body organ dysfunction of the mice is avoided, and simultaneously, kdf1 genes of tooth germ epithelial tissues can be specifically knocked out, so that conditions are created for researching functions of the Kdf1 genes.

Description

Kdf1 gene conditional knockout mouse model and construction method thereof

Technical Field

The invention relates to the technical field of genetic engineering mouse models, in particular to a Kdf1 gene conditional knockout mouse model and a construction method thereof.

Background

The human Kdf1 gene (keratinocyte differentiation factor 1) was found in 2013 to be located on human chromosome 1p36.11 and can encode 398 amino acid proteins. The mouse Kdf1 protein has 90% homology with human Kdf1 (C1 orf 172) protein, which lays a very favorable condition for researching the functions of human KDF1 genes by using a mouse model. Although the role of the Kdf1 gene in skin development of mice has been preliminarily determined, and it has been found that the Kdf1 protein is specifically expressed in the tooth germ epithelium and hardly expressed in the mesenchymal tissue, it is not clear about the developmental role of the Kdf1 gene in the tooth and oral mucosa epithelium and the like which are homologous to skin development. So far, only 3 Kdf1 gene mutations and congenital tooth deficiency reports exist, and the functions and pathogenicity of the Kdf1 genes need further research.

The construction of a Kdf1 gene-deficient mouse model is an important means for researching the functions and pathogenicity of the Kdf1 gene. In 2013, lee et al report a mouse model-shd mouse with Kdf1 systemic function defects for the first time, and the technical means are as follows: systemic induction of the Kdf1 gene with N-ethyl-N-nitrosourea (ENU) to appear T at the second intron>The G base is changed, so that the frame shift mutation of the extension of the amino acid sequence is caused by the frame change of the Kdf1 protein isomer 1, or the truncated mutation is caused by the in-frame deletion mutation of the Kdf1 protein isomer 2, and the functions of the mouse Kdf1 gene are further influenced. Subsequently, the investigator also used gene trapping techniques to isolate Kdf1 (1810019J 16Rik ^{tm1a(EUCOMM)Wtsi} ，Kdf1 ^GT ) Introducing mouse embryo stem cell to establish Kdf1 with Kdf1 gene base substitution mutation ^GT/GT Mice, the mouse modelThe systemic phenotype of the model was highly consistent with that of the shd mice, and it was further mutually demonstrated that developmental defects of both mice were caused by loss of Kdf1 gene function. However, the mouse model constructed in the above study is defective in function of the systemic Kdf1 gene, meaning that the Kdf1 gene expressed in different developmental tissues throughout the body of the mouse is lost, and therefore, the whole body tissue organ disorder of the mouse model causes perinatal death. The mouse model lacks the tissue specificity of gene knockout, and is difficult to accurately study the action and mechanism of Kdf1 genes in the development process of different tissues and organs. In addition, the problems that the gene mutation point is difficult to predict, the specific source cells cannot be knocked out specifically, the knocking-out rate is low, the knocking-out method is complex and the like exist in the gene mutation mouse model established by using the prior art such as the induction of medicines such as ENU and the like or the gene mutation mouse model established by trapping the vector based on the mouse embryonic stem cells. There is a need to construct a novel Kdf1 gene-deficient mouse model to meet the requirements of accurate research on the action mechanism of Kdf1 gene for regulating and controlling dental germ and oral epithelium.

Disclosure of Invention

The invention aims to provide a construction method of a Kdf1 gene conditional knockout mouse model, which aims to solve the technical problem that the Kdf1 gene defect mouse model in the prior art is difficult to realize accurate research on the action mechanism of Kdf1 regulation dental embryo and oral epithelium.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the construction method of the Kdf1 gene conditional knockout mouse model comprises the following steps in sequence:

s1: obtaining a donor vector: the donor vector comprises an upstream loxP site and a downstream loxP site; exon2 and exon 4 located in the Kdf1 gene are located between the upstream loxP site and the downstream loxP site;

s2: obtaining gRNA;

s3: obtaining a Kdf1 gene conditional knockout mouse model.

The proposal also provides a method for constructing a Kdf1 gene conditional knockout mouse model, which obtains the genotype of Kdf1 ^flox/flox Kdf1 group of (2)Due to conditional knockdown of the mouse model.

The principle and the advantages of the scheme are as follows:

the technical proposal utilizes the CRISPer-Cas9 technology to construct a conditional knockout mouse model of Kdf1 gene, and obtains F0 generation mouse containing target gene which can be conditionally knocked out by constructing a donor vector containing two loxP sites and then modifying the genome of a mouse fertilized egg by combining the CRISPer-Cas9 technology, and obtaining Kdf1 with stable shape through multiple-generation hybridization and breeding ^flox/flox And (3) a mouse. When using tissue specific Cre, such as K14-Cre mice or Pitx2-Cre mice with Kdf1 ^flox/flox The mice are mated, so that the gene functions of the mice can be comprehensively inactivated, and a stable and efficient gene knockout effect is achieved. Wherein Cre-loxP recombination is a mature technology in the prior art, is a recombinase technology of a specific site, can perform deletion, insertion, translocation and inversion on the specific site of DNA, and can modify DNA in cells by using the system aiming at specific cell types or adopting specific external stimulus.

In conclusion, the beneficial effects of the technical scheme are as follows:

(1) Kdf1 of the invention ^flox/flox The mouse model can be mated with Cre recombinase mice with various tissue sources, so that the mice with the Kdf1 genes knocked out by various tissue specificities can be constructed, and embryonic lethality caused by the systemic organ dysfunction of the mice can be avoided. On the other hand, the Kdf1 gene is only expressed in epithelial tissues such as tooth germ epithelium, and the Kdf1 gene of the epithelial tissues of the tooth germ is specifically knocked out, so that the accurate research of the action mechanism of the Kdf1 gene for regulating and controlling the tooth germ and the oral epithelium is facilitated.

(2) The Crisper-Cas9 technology is very simple and convenient to construct and use, has low cost, does not integrate exogenous DNA in the whole targeting process, and avoids the biosafety problem caused by the traditional transgene. On the basis of precisely knocking out genes in animal genome, the expression and interaction conditions of different genes and molecules are clarified, so that the functions of the genes and the regulation network are studied in depth.

(3) The Kdf1 gene has four exons (exon 1-4) and five transcripts, and the inventor has found in earlier research that the second exon to the fourth exon of the Kdf1 gene are taken as target fragments, and can knock out the whole coding sequence of the Kdf1 gene to completely inactivate the functions of the Kdf1 gene. In designing the loxP site, the insertion of the loxP site should avoid influencing the expression of the target gene (before mating with Cre tool mice), and in consideration of ensuring complete inactivation of Kdf1 gene after mating with Cre tool mice, the scheme selects to insert upstream loxP at a non-conservative site upstream of exon2 and downstream loxP at a non-conservative site downstream of exon 4, so as to avoid the influence of the insertion of loxP on the expression of the target gene.

Further, in S1, the donor vector includes an upstream loxP site located at 839-873bp upstream of exon2 of the Kdf1 gene and a downstream loxP site located at 560-594bp downstream of exon 4 of the Kdf1 gene.

In designing the loxP site, the insertion of the loxP site should avoid influencing the expression of the target gene (before mating with Cre tool mice), and in consideration of ensuring complete inactivation of Kdf1 gene after mating with Cre tool mice, the scheme selects a non-conservative site where upstream loxP is inserted at 839-873bp upstream of exon2 and downstream loxP is inserted at a non-conservative site where 560-594bp downstream of exon 4, so that the influence of the insertion of the loxP on the expression of the target gene is avoided. In this protocol, knockout of exon2-4 is the optimal choice, and can affect all transcripts (five transcripts all affected). Furthermore, only by inserting loxp into these two specific non-conserved sites, all knockout of exon2-4 can be achieved without affecting the function of the gene before conditional knockout, and without affecting correct splicing and mouse survival.

Further, in S1, the donor vector comprises a 5 'homology arm region with a sequence shown as SEQ ID NO.2, an upstream loxP site with a sequence shown as SEQ ID NO.4, selective knocking-out with a sequence shown as SEQ ID NO.1, a downstream loxP site with a sequence shown as SEQ ID NO.4 and a 3' homology arm region with a sequence shown as SEQ ID NO.3, which are sequentially arranged from the 5 'end to the 3' end.

By adopting the technical scheme, the design of the donor vector can also avoid the problems that arm and cKO contain sequences which are completely matched with the gRNA, so that the gRNA can possibly generate off-target on the donor vector or homologous recombination repair site. The above design also avoids insertion of loxP site and should avoid affecting expression of target gene.

Further, in S2, the gRNA includes gRNA1 having a gene sequence shown as SEQ ID NO.5, gRNA2 having a gene sequence shown as SEQ ID NO.6, gRNA3 having a gene sequence shown as SEQ ID NO.7, and gRNA4 having a gene sequence shown as SEQ ID NO. 8.

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

Further, the step S3 comprises a microinjection step, a positive F0 mouse obtaining step, a F1 generation mouse obtaining step and a F2 generation mouse obtaining step; in the microinjection step, the Donor vector, gRNA and Cas9 protein are co-injected into the mouse fertilized egg; returning the microinjected fertilized eggs to the oviduct of the surrogate mice; the mice are identified and screened after birth to obtain positive F0 mice. And transferring the exogenous gene into fertilized eggs by microinjection to obtain transgenic mice.

Further, in the step of obtaining the positive F0 mice, the fertilized eggs after microinjection are returned to the oviduct of the surrogate mice; the mice are subjected to PCR and sequencing identification screening after birth, and positive F0 mice are obtained. In the microinjection process, 550 fertilized eggs are injected in total before and after the microinjection process and returned to the oviduct of the surrogate mother, 64F 0 mice are born in total, the birth rate is 12%,5 fixed-point positive F0 mice are 7.8%, and the positive rate is 100%.

Further, in the step of obtaining F1 mice, sexually mature positive F0 mice were bred with wild mice, and the genotype was selected as Kdf1 by PCR ^flox/+ F1 generation mice of (C). Genetically stable transgenic mouse offspring were obtained by crossing.

Further, in the step of obtaining F2 mice, the genotype was Kdf1 ^flox/+ The F1 generation mice of (2) are inbred for breeding generation, and the genotype is Kdf1 is screened out by a PCR method ^flox/flox F2 generation mice of (c). The homozygous F2 generation mice are obtained through inbreeding and can be used for subsequent conditional gene migration experiments。

Drawings

FIG. 1 shows the sequence (first part) of the donor vector of example 1 of the present invention.

FIG. 2 shows the sequence (second part) of the donor vector of example 1 of the present invention.

FIG. 3 shows the sequence of the donor vector of example 1 (third part) of the present invention.

FIG. 4 shows the sequence of the donor vector of example 1 (fourth part) of the present invention.

FIG. 5 shows the sequence of the donor vector of example 1 (fifth part) of the present invention.

FIG. 6 is a schematic diagram showing the structure of the donor vector, the wild-type allele of Kdf1 gene, the mutant allele containing cKO and the mutant allele after Cre recombination in example 1 of the present invention.

FIG. 7 is a chromosomal localization analysis of gRNA1 of example 1 of the present invention.

FIG. 8 is a chromosomal localization analysis of gRNA2 of example 1 of the present invention.

FIG. 9 is a chromosomal localization analysis of gRNA3 of example 1 of the present invention.

FIG. 10 is a chromosomal localization analysis of gRNA4 of example 1 of the present invention.

FIG. 11 is a off-target analysis of gRNA1 of example 1 of the present invention.

FIG. 12 is a off-target analysis of gRNA2 of example 1 of the present invention.

FIG. 13 is a off-target analysis of gRNA3 of example 1 of the present invention.

FIG. 14 is a off-target analysis of gRNA4 of example 1 of the present invention.

FIG. 15 is a schematic representation of the RCR and sequencing primer binding sites (F1 generation) of example 2 of the present invention.

FIG. 16 is an electrophoresis chart (PCR primer set 1) of RCR verification of example 2 of the present invention.

FIG. 17 is a RCR of example 2 of the present invention electrophoresis pattern of the verification (PCR primer pair 2).

FIG. 18 is a diagram of example 2 of the present invention sequencing result graph (F1 generation).

FIG. 19 is a schematic representation of the binding sites of the RCR primers of example 2 of this invention (first identification of F0 generation).

FIG. 20 is a first identification electrophoresis pattern (primer F5R 4) of example 2 of the present invention.

FIG. 21 is a first identification electrophoresis pattern (primer F2R 3) of example 2 of the present invention.

FIG. 22 is a second identification electrophoresis pattern (primer F1R 1) of example 2 of the present invention.

FIG. 23 is a second identification electrophoresis pattern (primer F2R 2) of example 2 of the present invention.

FIG. 24 is an electrophoresis chart of F2 generation identification in example 2 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments, but the embodiments of the present invention are not limited thereto. The technical means used for the following implementation are, unless otherwise indicated, conventional means well known to those skilled in the art: the materials, reagents, and the like used are all commercially available. Mouse genome extraction in experimental examples was performed using TaKaRa kit (TaKaRa MiniBEST Universal Genomic DNA Extraction kit) and rat tails were taken for genome extraction.

Example 1: vector and gRNA (guide-RNA) design and construction

Kdf1 gene has a accession number NM-001083916.1, kdf1 ^flox/flox The construction process of the mouse model comprises the following steps:

the method mainly comprises the steps of designing a gRNA sequence and a targeting vector scheme; the In-Fusion technique was used to construct a donor vector (targeting vector) comprising loxP sites, and verifying the targeting vector by enzyme digestion, PCR and sequencing.

The sequence of the donor vector is shown in FIGS. 1-5, and the donor vector includes a 5 'homology arm region (5' arm), an upstream loxP site, a selective knock-out (cKO), a downstream loxP site, and a3 'homology arm region (3' arm). The light-colored region in FIGS. 1 to 5 is a cKO region (SEQ ID NO. 1), and the single underlined region in the light-colored region is an exon (exon 2-4 in order from front to back); the wavy line marked region in the dark part of the figure is 5'arm (SEQ ID NO. 2) and 3' arm (SEQ ID NO. 3), and the double underlined region is two loxP sites (SEQ ID NO. 4). The Kdf1 gene has four exons (exon 1-4) and five transcripts, and the inventor has found in earlier research that the second exon to the fourth exon of the Kdf1 gene are taken as target fragments, and can knock out the whole coding sequence of the Kdf1 gene to completely inactivate the functions of the Kdf1 gene. In designing the loxP site, the insertion of the loxP site should avoid affecting the expression of the target gene (before mating with Cre tool mice), and considering ensuring complete inactivation of Kdf1 gene after mating with Cre tool mice, the present scheme selects a non-conserved site where upstream loxP is inserted at 839-873bp upstream of exon2 and a non-conserved site where downstream loxP is inserted at 560-594bp downstream of exon 4, thereby avoiding the influence of the insertion of loxP on the expression of the target gene (see FIG. 6 and FIGS. 1-5). In the technical scheme, the knockout of exon2-4 is the optimal choice, and if only exon2 or exon2 and 3 are knocked out, partial gene coding regions still remain, so that the complete inactivation of Kdf1 gene functions cannot be realized. Furthermore, only by inserting loxp into these two specific non-conserved sites, all knockout of exon2-4 can be achieved without affecting the function of the gene before conditional knockout, and without affecting correct splicing and mouse survival. The loxp insertion site search also needs to take into account the off-target scoring of the gRNA, i.e. the validation of the loxp insertion site needs to be considered according to the specifics of the gRNA design. The design of the donor vector can also avoid the problems that arm and cKO contain sequences which are completely matched with the gRNA, so that the gRNA can not target off the donor vector or homologous recombination repair site. The inventors have tried other loxp insertion sites, none of which meets the above requirements, and have resulted in experimental results of the birth rate, the positive rate, and the survival rate of the site-specific positive F0 mice being far lower than those of example 2, and the growth state of the site-specific positive F0 mice being not ideal. Only the non-conserved sites at 839-873bp upstream of exon2 and the non-conserved sites at 560-594bp downstream of exon 4 can meet the requirements of the technical scheme. Confirmation of loxp insertion site is a result of extensive experimental study, and it is difficult to evaluate the effect of loxp insertion site selection before the experiment is actually performed.

gRNA target sequence:

gRNA1 (paired with the antisense strand of the gene): 5' -GGACGCCTCGTCTGCTCATAGGG-3', (SEQ ID NO.5, at chr4:133527089-133527111, FIG. 7, reference database: mus) musculus GRCm38/mm10)；

gRNA2 (paired with the sense strand of the gene): 5' -CTGCTGTATTATCCGGGTCTAGG-3', (SEQ ID NO.6, at chr4:133531335-133531357, FIG. 8).

In selecting a gRNA target sequence, the inventors tried a variety of target sequences, including also the following two sequences:

gRNA3 (paired with the sense strand of the gene): 5' -GAGTGCCCTATGAGCAGACGAGG-3', (SEQ ID No.7, at chr4:133527084-133527106, fig. 9);

gRNA4 (paired with the sense strand of the gene): 5' -TGGGTGCTGCTGTATTATCCGGG-3', (SEQ ID NO.8, at chr4:133531329-133531351, FIG. 10).

Results of off-target analysis of grnas 1-4 see fig. 11-14, with grnas 1 and 2 being optimal grnas.

Example 2: microinjection, identification of F0 mice, cultivation and screening of F1 mice

This step essentially involves co-injecting the Donor vector, gRNA (gRNA 1 and gRNA 2) and Cas9 protein (available from New England Biolabs company under accession number M0646M) into fertilized eggs by conventional means of the prior art. More specifically, tube 1 solution (mixture of gRNA and Cas9 protein obtained) was formulated: 0.8uL 100pmol/uL CrRNA (0.4ul+0.4ul) was added to 5.2uL RNase-free water, followed by incubation for 5min with 0.6uL 100pmol/uL TracRNA, followed by incubation for 10min with 0.2uL Cas9 protein (NEB, cat# M0646M) to give tube 1 solution (gRNA 1 and gRNA 2); preparing a tube 2 solution: donor plasmid with final concentration of 15 ng/uL; equal volumes of the tube 1 solution and the tube 2 solution were mixed to obtain an RNP injection complex, and fertilized eggs of mice were injected. Wherein, crRNA is CRISPR RNA, and tracrRNA (trans-activating crRNA) is transactivating crRNA; in the CRISPR/Cas9 system, crRNA can combine with tracrRNA to form gRNA (guide RNA), then Cas9 protein is precisely positioned to a target site with the assistance of gRNA, and finally, precise editing of DNA double strand is completed. Both tracrRNA and crRNA can be designed and obtained by conventional means in the art after the identification of the target sequence of the gRNA, and can be commercially commissioned for synthesis by related biotechnology companies. The fertilized eggs after microinjection are returned to the oviduct of the surrogate mice; the mice were identified by PCR and sequencing after birth to obtain positive F0 mice.

The sexually mature positive F0 mice are respectively matched with wild mice for reproduction generation, and the genotype of Kdf1 is screened out by a PCR experimental method ^flox/+ The primer sequences of the F1 generation mice of (2) are as follows:

PCR primer pair 1:

5' arm forward primer (F1): 5'-TCCTATTGACCTGCCCCTTAGCG-3';

3' loxP reverse primer (R1): 5'-GTGGATTCGGACCAGTCTGA-3';

PCR primer pair 2:

5' loxP forward primer (F2): 5'-GTGCCCTATGAGCAGACATAACT-3';

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

Schematic representation of the amplification sites of PCR primer pair 1 and PCR primer pair 2 is shown in FIG. 15. The result of electrophoresis after amplification is shown in FIGS. 16 and 17. After constructing F1 flox heterozygous mice, the primers F1R1 and F2R2 are used for verification, and the primer pair can cover the whole area, so that whether the complete recombination is correct or not can be identified (the possibility that the complete recombination is not formed, and the recombination is formed but not complete exists). In addition, sequencing (F1 generation sequencing results see FIG. 18) and southern verification were performed to further confirm that the sequence of interest was fully correctly recombined. The above experimental results have verified that the mice were successfully constructed, but if the subsequent breeding is performed completely according to this procedure, the process is very cumbersome. Therefore, a simpler verification and authentication party is adopted later, and only a part is required to be verified to confirm whether the target sequence exists or not (see later for details).

Simultaneously performing sequencing verification, wherein the sequencing primer comprises:

5' sequencing primer (F3): 5'-CTCCTCTGACCTCCGTGGGC-3';

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

Typical sequencing results referring to fig. 14, sequencing results and electrophoresis results demonstrate successful construction of mutant alleles containing cKO.

During microinjection, total of front and rear550 fertilized eggs are injected and returned to the oviduct of the surrogate mother, 64F 0 mice are born in total, the birth rate is 12%,5 fixed-point positive F0 mice are positioned, the positive rate is 7.8%, and the survival rate is 100%. Wherein two determinations were made for the F0 mice. The schematic diagram of the amplification sites identified at the first time is shown in FIG. 19, and the schematic diagram of the amplification sites identified at the second time is shown in FIG. 15 (the F1 identification of the primer is F1R1 and F2R2 primers). The positive F0 mice can be successfully matched with wild mice to obtain offspring with genotype Kdf1 ^flox/+ F1 generation mice of (C). The primer pairs used for the primary identification were F2 and R3 (336 bp for amplification of the target allele) and F5 and R4 (216 bp for amplification of the target allele, 142bp for the wild-type allele, see below).

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

F0 primary identification result (primer F5R 4) is shown in FIG. 20; f0 primary identification (primer F2R 3) is shown in fig. 21 (total test of 44 samples, here selected as an example of a partial sample electropherogram). The secondary identification result of F0 (primer F1R 1) is shown in FIG. 22; the primary F0 identification result (primer F2R 2) is shown in FIG. 23 (sample No. 42/14/21/23/44/2/3/4 is selected).

F1 generation mice are continuously matched with a first generation (inbreeding), and a PCR experimental method is used for screening out the genotype Kdf1 ^flox/flox The primer sequences of F2 generation mice of (2) are as follows:

5' forward primer (F5): 5'-AGTTTCCTAATGGACACTACAGGAC-3';

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

Electrophoresis results show that Kdf1 ^flox/flox Is a 216bp electrophoresis band; kdf1 ^flox/+ Two bands of 216bp and 142 bp; kdf1 ^+/+ Is a 142bp electrophoresis band. F2 identification results are shown in fig. 24.

When the scheme is used for constructing a conditional knockout mouse model of the Kdf1 gene by utilizing the CRISPer-Cas9 technology, exons of all coding regions of the Kdf1 gene of the mouse model can be knocked out, so that the gene functions are completely inactivated, and a stable and efficient gene knockout effect is achieved. Compared with the traditional method, the construction process is simplified, the cost is lower, the period is short, and the whole exogenous DNA does not exist in the whole targeting processAnd the biological safety problem caused by the traditional transgenesis is avoided. When using tissue specific Cre, such as K14-Cre mice or Pitx2-Cre mice with Kdf1 ^flox/flox The conditional gene knockout mice generated by mating the mice have tissue-specific knockout, which is beneficial to realizing the accurate research of the action mechanism of Kdf1 gene in regulating and controlling dental germ and oral epithelium.

The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (10)

1. The construction method of the Kdf1 gene conditional knockout mouse model is characterized by comprising the following steps of:

s1: obtaining a donor vector: the donor vector comprises an upstream loxP site and a downstream loxP site; exon2 and exon 4 located in the Kdf1 gene are located between the upstream loxP site and the downstream loxP site;

s2: obtaining gRNA;

s3: obtaining a Kdf1 gene conditional knockout mouse model.

2. The method according to claim 1, wherein in S1, the donor vector comprises an upstream loxP site located at 839-873bp upstream of exon2 of Kdf1 gene and a downstream loxP site located at 560-594bp downstream of exon 4 of Kdf1 gene.

3. The method according to claim 2, wherein in S1, the donor vector comprises a 5 'homology arm region with a sequence shown in SEQ ID NO.2, an upstream loxP site with a sequence shown in SEQ ID NO.4, a selective knock-out with a sequence shown in SEQ ID NO.1, a downstream loxP site with a sequence shown in SEQ ID NO.4 and a 3' homology arm region with a sequence shown in SEQ ID NO.3, which are sequentially arranged from the 5 'end to the 3' end.

4. The method for constructing a model of a Kdf1 gene conditional knockout mouse according to claim 3, wherein in S2, the gRNA includes gRNA1 having a gene sequence shown in SEQ ID NO.5, gRNA2 having a gene sequence shown in SEQ ID NO.6, gRNA3 having a gene sequence shown in SEQ ID NO.7, and gRNA4 having a gene sequence shown in SEQ ID NO. 8.

5. The method for constructing a model of a Kdf1 gene conditional knockout mouse according to claim 4, wherein in S2, the gRNA consists of gRNA1 with a gene sequence shown as SEQ ID NO.5 and gRNA2 with a gene sequence shown as SEQ ID NO. 6.

6. The method according to claim 5, wherein the S3 comprises a microinjection step, a positive F0 mouse obtaining step, a F1-generation mouse obtaining step, and a F2-generation mouse obtaining step; in the microinjection step, the Donor vector, gRNA and Cas9 protein are co-injected into the mouse fertilized eggs.

7. The method for constructing a model of a Kdf1 gene conditional knockout mouse according to claim 6, wherein in the step of obtaining positive F0 mice, the microinjected fertilized eggs are returned to oviducts of surrogate mice; the mice are subjected to PCR and sequencing identification screening after birth, and positive F0 mice are obtained.

8. The method for constructing a model of a Kdf1 gene conditional knockout mouse according to claim 7, wherein in the step of obtaining the F1-generation mouse, sexually mature positive F0 mice are bred with wild-type miceFirst generation, the genotype of Kdf1 is screened out by PCR method ^flox/+ F1 generation mice of (C).

9. The method for constructing a model of a Kdf1 gene conditional knockout mouse according to claim 8, wherein in the step of obtaining the F2-generation mouse, the genotype is Kdf1 ^flox/+ The F1 generation mice of (2) are inbred for breeding generation, and the genotype is Kdf1 is screened out by a PCR method ^flox/flox F2 generation mice of (c).

10. A method for constructing a mouse model of conditional knock-out of a kd 1 gene according to any one of claims 1 to 9, which gives a genotype of kd 1 ^flox/flox A conditional knockout mouse model of the Kdf1 gene of (2).

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