CN112899279A - Method for constructing Fzd6 gene knockout mouse model and application - Google Patents
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Abstract
The invention discloses a method for constructing an Fzd6 gene knockout mouse model and application thereof, the method uses CRISPR/Cas9 gene editing technology to target the 4 th exon of the Fzd6 gene, designing and synthesizing sgRNA at corresponding positions, then microinjecting the sgRNA/Cas9 transcribed in vitro into fertilized eggs of mice together, transplanting the fertilized eggs into the uterus of a pseudopregnant female mouse to obtain an F0 generation mouse, then identifying positive mice by PCR and sequencing, backcrossing with wild background mice to obtain heterozygote mice of single genotype, then cross breeding between the heterozygote mice to obtain homozygote mice, that is, the sequence of the constructed Fzd6 gene knockout mouse is subjected to sequencing to delete 5 bases at a target position and cause frame shift mutation, the model mouse can be used for researching the function of FZD6 in the involved cancers, neurodevelopmental, hematopathy and mental diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for constructing an Fzd6 gene knockout mouse model and application thereof.
Background
The frizzled (fzd) family of proteins is a specific class of receptors in the Wnt/β -catenin signaling pathway, a class of transmembrane proteins, located on the plasma membrane of cells. These receptors activate the Wnt/β -catenin signaling pathway by binding to Wnt signaling molecules, thereby activating downstream target genes and functioning in the body. In mammals, the FZD protein family comprises a total of 10 members, is evolutionarily conserved, is widely expressed in cells, and has a conserved structure in different animals and tissues, namely: it contains a cysteine-rich crd (cysteine rich domain) domain at the N-terminus and a conserved ktxxvw domain at the C-terminus. It has been found that members of the FZD protein family play a very important role in animal development and in cancer.
FZD6 is a member of the FZD protein family, the mouse Fzd6 gene is located on chromosome 15 (39,006,034-39,038,188), the gene has a total length of 32044bp, and comprises 7 exons and 5 transcripts, wherein the transcript 2 is the longest and is 4325bp, and 709 amino acids are coded in total. The structure of the human FZD6 gene is similar to that of a mouse, has high homology and conservation, and has 8 Single Nucleotide Polymorphism (SNP) sites, wherein 4 SNPs are missense mutation SNPs, and two SNPs are positioned on a fourth exon. FZD6 has been reported to play a very important role in neural tube malformations, hair development, hematological diseases, psychiatric diseases, and cancer, and therefore it is very important to construct a mouse model of this gene knockout.
The commonly used method for constructing knockout mice at present comprises: transcription activator-like effector nuclease (TALEN) technology, Zinc Finger Nuclease (ZFN) technology, Embryonic stem cell (ES) targeting technology and the like, however, the construction period of the methods is long, the cost is high, and certain off-target problems exist. However, the existing animal models for knocking out the Fzd6 gene are mainly knocked out by a lentivirus injection method or the gene editing technology at a specific position of the brain, a model for knocking down by a small interfering rna (sirna) method is more used, and a mouse model for knocking out the Fzd6 gene by a newer technology is not available.
Disclosure of Invention
In view of this, an object of the embodiments of the present invention is to provide a method for constructing an Fzd6 gene knockout mouse model based on the latest gene editing technology and an application thereof, so as to solve the problems of long process cycle, high cost and complicated operation of the currently commonly used process for constructing the gene knockout mouse, and provide a mouse model for researching the biological function of Fzd6 in diseases in which Fzd6 participates.
According to a first aspect of embodiments of the present invention, there is provided a method of constructing an Fzd6 gene knockout mouse model, comprising:
(1) determining a specific target site of a gene to be knocked out of a mouse, and designing a sgRNA sequence aiming at a fourth exon of a mouse Fzd6 gene, wherein the specific sgRNA sequence is SEQ ID NO. 1;
(2) constructing an sgRNA/Cas9 expression vector by using the sgRNA;
(3) after the sgRNA/Cas9 expression vector is linearized and purified, sgRNA and Cas9mRNA are synthesized through in vitro transcription;
(4) microinjecting the sgRNA and Cas9mRNA into mouse fertilized eggs;
(5) transplanting the fertilized eggs surviving after injection into a pseudopregnant mother mouse, wherein the born mouse is an F0 generation mouse, and the wild mouse or a positive heterozygote mouse is confirmed through PCR reaction and Sanger sequencing;
(6) backcrossing the identified F0 generation wild-type mice and the positive heterozygote mice to obtain heterozygote mice with a single genotype, and then carrying out hybridization breeding among the heterozygote mice to obtain homozygote individuals, namely the constructed Fzd6 gene knockout mouse model.
Further, constructing an sgRNA/Cas9 expression vector in (2), specifically:
(1) designing specific primers for sgRNA sequences, wherein the specific primers comprise a sgRNA forward primer and a sgRNA reverse complementary primer;
(2) annealing the designed sgRNA into double chains in a gradient cooling mode, connecting the double chains with plasmids, converting, coating a plate, then placing the double chains in a bacterial incubator at 37 ℃ for overnight incubation, picking and inoculating the single clones in LB culture solution the next day, culturing the single clones in a constant temperature shaking table at 37 ℃ for overnight, then collecting bacterial solution for plasmid extraction, and simultaneously carrying out PCR identification on positive clones.
Further, the sequence of the sgRNA forward primer is SEQ ID No.2, and the sequence of the sgRNA reverse complementary primer is SEQ ID No. 3.
According to a second aspect of the embodiments of the present invention, there is provided an Fzd6 gene knockout mouse model constructed by the method described above.
According to a third aspect of embodiments of the present invention there is provided the use of a Fzd6 gene knockout mouse model that can be used to study the role of Fzd6 in cancers, neurodevelopmental, hematologic and psychiatric disorders in which it is involved.
According to a fourth aspect of the embodiments of the present invention, there is provided a kit for constructing an Fzd6 gene knockout mouse model, comprising a sgRNA having a sequence of SEQ ID No. 1.
Further, Cas9mRNA was also contained.
According to the technical scheme, the invention has the following beneficial effects: compared with the common technology, the technology adopted by the invention is simple, efficient, quick and low in cost, and can cause base deletion and frameshift mutation of the Fzd6 gene. Meanwhile, the mouse model provided by the invention lays a good foundation for further researching the function of the FZD6 in the cancers, the neural development, the blood diseases and the mental diseases involved in the FZD 6.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
FIG. 1 is a schematic diagram of the knockout strategy of Fzd6 gene in the example of the present invention.
FIG. 2 is a schematic diagram of the PCR identification strategy in the example of the present invention.
FIG. 3 is a diagram showing the sequence analysis of the knockout region of the Fzd6 gene knockout mouse in the example of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples in conjunction with the accompanying drawings.
The embodiment of the invention provides a mouse model for knocking out Fzd6 gene based on the latest CRISPR/Cas9(Clustered regulated interleaved short palindromic repeats/CRISPR-associated nuclear 9) gene editing technology, which is a third generation gene editing technology after ZFN, TALENs and other technologies, the mouse model is a mouse with knocked-out Fzd6 gene, the strain of the mouse is C57BL/6J, the name of the knocked-out gene (NCBI number) is Fzd6(14368), and the selected transcript (Ensembl number) is Fzd6-202(ENSMUST 00000179165.8).
The embodiment of the invention provides a method for constructing an Fzd6 gene knockout mouse model based on CRISPR/Cas9 gene editing technology, wherein a knockout strategy is shown in figure 1 and comprises the following steps:
firstly, the target site of the gene to be knocked out is determined to be located in the fourth exon of the Fzd6 gene, and then a sequence-specific sgRNA and a corresponding primer are designed. The sequence of the sgRNA is SEQ ID NO.1, the sequence of the sgRNA forward primer is SEQ ID NO.2, and the sequence of the sgRNA reverse complementary primer is SEQ ID NO. 3.
| Name (R) | Serial number | Sequence of |
| sgRNA | SEQ ID NO.1 | CACCAAAATCCAATGTCTCT |
| Fzd6-ko-TF1 | SEQ ID NO.2 | TCTGTGAATGCAGCAAAGTCATGG |
| Fzd6-ko-TR1 | SEQ ID NO.3 | GTCTCTCTGGGTATCTGAATCGTC |
Then, an expression vector of sgRNA/Cas9 is constructed, and the designed sgRNA is annealed into double strands in a gradient cooling mode and then connected with a plasmid. Transformation and LB plate plating were carried out using DH 5. alpha. E.coli as a vector, and the plates were incubated overnight in a bacterial incubator at 37 ℃. Approximately 10 single clones on the plate were then picked in a sterile operating station, inoculated in LB broth and cultured overnight in a constant temperature shaker at 37 ℃ and 160 rpm. Then collecting bacterial liquid to extract plasmid, and simultaneously carrying out PCR identification positive cloning.
Then, the expression vector of sgRNA/Cas9 was linearized and purified as a template according to megashort scriptTMThe T7 Transcription Kit synthesized sgRNA and Cas9mRNA in vitro.
Subsequently, the transcribed sgRNA and Cas9mRNA were injected into mouse zygotes together by microinjection and transplanted into the uterus of pseudopregnant females, i.e., mice born for F0 generation, and then genotyped by PCR and sequencing, as follows:
collecting the mouse tail, extracting DNA by using an animal tissue DNA extraction kit, then carrying out PCR reaction by using the primers, and identifying according to a PCR identification strategy diagram shown in figure 2, wherein the reaction system is as follows:
| composition (I) | Volume (μ l) |
| 2×Taq Master Mix，Dye Plus | 12.5 |
| Upstream primer Fzd6-ko-TF1(10 pmol/. mu.l) | 1 |
| Downstream primer Fzd6-ko-TR1(10 pmol/. mu.l) | 1 |
| DNA template (. apprxeq.100 ng/. mu.l) | 1 |
| ddH2O | 9.5 |
The reaction conditions for PCR were as follows:
and performing 2% agarose gel electrophoresis on the PCR product, then performing tapping recovery, purifying the PCR product, and identifying whether the sequence has deletion by Sanger sequencing, wherein the sequencing primer sequence is SEQ ID NO. 3.
And (3) carrying out backcross on the F0 mouse identified as positive and a wild background mouse to obtain a heterozygote mouse with a single genotype, and then carrying out hybridization breeding among the heterozygote mice to obtain a homozygote individual, namely the constructed Fzd6 gene knockout mouse model. The identification method is the same as the PCR and sequencing method.
As shown in FIG. 3, the deletion of 5 bases in the third exon of the Fzd6 gene (sequence: ATGTC) results in the frame shift mutation of the downstream gene sequence, which indicates that the Fzd6 gene knockout mouse is successfully obtained and the model construction is successful.
The mouse model constructed by the invention can be used for researching the function of the FZD6 in the cancers, the neural development, the blood diseases and the mental diseases.
The embodiment of the invention also provides a kit for constructing an Fzd6 gene knockout mouse model, which contains sgRNA, wherein the sequence of the sgRNA is SEQ ID NO. 1. Further, Cas9mRNA was also contained.
Finally, it should be noted that: these embodiments are merely illustrative of the present invention and do not limit the scope of the present invention. In addition, other variations and modifications will be apparent to persons skilled in the art based on the foregoing description. It is not necessary or necessary to exhaustively enumerate all embodiments herein, and obvious variations or modifications can be made without departing from the scope of the invention.
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<120> method for constructing Fzd6 gene knockout mouse model and application
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Claims (7)
1. A method for constructing an Fzd6 gene knockout mouse model is characterized by comprising the following steps:
(1) determining a specific target site of a gene to be knocked out of a mouse, and designing a sgRNA sequence aiming at a fourth exon of a mouse Fzd6 gene, wherein the specific sgRNA sequence is SEQ ID NO. 1;
(2) constructing an sgRNA/Cas9 expression vector by using the sgRNA;
(3) after the sgRNA/Cas9 expression vector is linearized and purified, sgRNA and Cas9mRNA are synthesized through in vitro transcription;
(4) microinjecting the sgRNA and Cas9mRNA into mouse fertilized eggs;
(5) transplanting the fertilized eggs surviving after injection into a pseudopregnant mother mouse, wherein the born mouse is an F0 generation mouse, and the wild mouse or a positive heterozygote mouse is confirmed through PCR reaction and Sanger sequencing;
(6) backcrossing the identified F0 generation wild-type mice and the positive heterozygote mice to obtain heterozygote mice with a single genotype, and then carrying out hybridization breeding among the heterozygote mice to obtain homozygote individuals, namely the constructed Fzd6 gene knockout mouse model.
2. The method of claim 1, wherein the sgRNA/Cas9 expression vector constructed in (2) is specifically:
(1) designing specific primers for sgRNA sequences, wherein the specific primers comprise a sgRNA forward primer and a sgRNA reverse complementary primer;
(2) annealing the designed sgRNA into double chains in a gradient cooling mode, connecting the double chains with plasmids, converting, coating a plate, then placing the double chains in a bacterial incubator at 37 ℃ for overnight incubation, picking and inoculating the single clones in LB culture solution the next day, culturing the single clones in a constant temperature shaking table at 37 ℃ for overnight, then collecting bacterial solution for plasmid extraction, and simultaneously carrying out PCR identification on positive clones.
3. The method of claim 2, wherein the sgRNA forward primer sequence is SEQ ID No.2 and the sgRNA reverse complement primer sequence is SEQ ID No. 3.
4. An Fzd6 knock-out mouse model constructed by the method of any one of claims 1-3.
Use of a Fzd6 gene knockout mouse model for studying the role of Fzd6 in cancers, neurodevelopmental, hematologic and psychiatric disorders in which it is involved.
6. The kit for constructing the Fzd6 gene knockout mouse model is characterized by containing sgRNA, wherein the sequence of the sgRNA is SEQ ID NO. 1.
7. The kit of claim 6, further comprising Cas9 mRNA.
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