CN117467710A - Construction method and application of Ccdc182 gene knockout mouse model - Google Patents
Construction method and application of Ccdc182 gene knockout mouse model Download PDFInfo
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- CN117467710A CN117467710A CN202311456414.0A CN202311456414A CN117467710A CN 117467710 A CN117467710 A CN 117467710A CN 202311456414 A CN202311456414 A CN 202311456414A CN 117467710 A CN117467710 A CN 117467710A
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Abstract
The application belongs to the field of animal models, and particularly relates to a construction method and application of a Ccdc182 gene knockout mouse model. The application provides a construction method of a Ccdc182 gene knockout mouse model, which designs a sgRNA sequence of a target site and can efficiently and accurately construct the Ccdc182 gene knockout mouse model for subsequent functional research by using a CRISPR/Cas9 technology. The CRISPR/Cas9 technology is used for constructing a Ccdc182 (NM_ 028859.1) gene knockout mouse animal model, and a good foundation is laid for further researching the function of the Ccdc182 gene and the genetic research of the Ccdc182 gene in the oligospermia of mice or humans.
Description
Technical Field
The application belongs to the field of animal models, and particularly relates to a construction method and application of a Ccdc182 gene knockout mouse model.
Background
Semen abnormality is classified into semen abnormality, which refers to the number of semen, color abnormality, abnormality of the cytoplasm, and sperm abnormality, which refers to the number of sperm, abnormality of the cytoplasm, and the like.
Causes of oligospermia include the following 7 aspects: 1. when varicocele occurs, the local temperature of testis is raised, and the vasoactive substances are increased, thereby affecting the function of testis to produce sperm. But the extent of varicocele is not proportional to sperm quality. 2. Cryptorchid is one of the important reasons affecting semen quality. About 60% of patients with unilateral cryptorchid are sterile, so if sperm density is low and cryptorchid exists, early treatment is necessary. 3. Chronic infection of the genital tract with accessory genital glands can affect various assay indicators in semen. 4. Autoimmune reproductive immunology studies have found that male autoimmunity can affect fertility and anti-sperm antibodies can affect sperm production and transport. 5. Normal spermatogenic function in men with endocrine abnormalities depends on the normal hypothalamic-pituitary-gonadal shaft function, wherein any one of the disorders affects the spermatogenic function, and other diseases such as thyroid and adrenal gland diseases affect the gonadal function to cause oligospermia. 6. Chromosome abnormal chromosome aberration has a serious influence on sperm density, motility and morphology. 7. Other scrotum temperatures are too high, radiation injury, chemical drugs and drug effects can cause oligospermia.
Male sperm abnormalities are an important etiology of infertility together. To provide a new diagnosis and treatment concept for male infertility for more fully and detailed understanding of spermatogenesis, related pathogenic genes need to be mined and functionally explored. The Ccdc182 gene (coiled-coil domain containing 182), which is located on mouse chromosome 11 in NC-000077.7 (88184869-88186087), has 1 transcript in mouse (NM-028859.1), full length 1219bp, contains 1 exon, and the encoded protein contains 153 amino acid residues.
At present, no relevant report on mutation of a Ccdc182 gene knockout mouse model is available for researching male infertility patients.
Disclosure of Invention
Based on the above, an embodiment of the present application provides a construction method and application of a Ccdc182 knockout mouse model, which are used for researching mutation of male sterile patients.
A kit for constructing a Ccdc182 gene knockout mouse model, the kit comprising sgRNA1 and sgRNA2.
The sequence of the sgRNA1 is shown as SEQ ID NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2.
In one embodiment, corresponding detection primers are also included.
The sequence of the detection primer is shown as SEQ ID NO. 3-SEQ ID NO. 4.
In one embodiment, the kit further comprises Cas9 mRNA.
In one embodiment, the kit further comprises Taq DNA polymerase, dNTPs and Mg 2+ One or more of the following.
A construction method of a Ccdc182 gene knockout mouse model comprises the following steps:
(1) Obtaining sgRNA1 and sgRNA2 of a targeted mouse Ccdc182 gene;
the sequence of the sgRNA1 is shown as SEQ.ID.NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2.
(2) And constructing an sgRNA/Cas9 expression vector by adopting the sgRNA1 and the sgRNA2, and performing in vitro transcription to prepare mRNA.
(3) The mRNA was introduced into pseudopregnant female mice to prepare F0 mice.
(4) And (3) screening a mice genetic line with stable Ccdc182 gene knockout from the F0 generation mice, hybridizing a positive F0 generation mouse with a wild type mouse to obtain an F1 generation heterozygote mouse, and mating the F1 generation heterozygote mouse to obtain an F2 generation Ccdc182 gene knockout homozygous mouse with stable genetic variation.
In one embodiment, the mouse strain is selected from C57BL/6.
In one example, positive mice were identified by PCR, and the detection primers used in the screening by PCR were shown in SEQ ID NO.3 and SEQ ID NO. 4.
In one embodiment, the amplification procedure for screening by PCR method is: pre-denaturing the reaction mixture at 95℃for 3min; carrying out a denaturation procedure at 95 ℃ for 30s, an annealing procedure at 60 ℃ for 60s, and an extension procedure at 72 ℃ for 60s, wherein the denaturation is carried out for 34 cycles; finally, thoroughly extending the procedure and reacting for 5min at 72 ℃, wherein the reaction is stored at 4 ℃ for short term and-20 ℃ for long term after the reaction is finished.
The application provides the application of the kit or the Ccdc182 gene knockout mouse model obtained by the construction method in drug screening.
In one embodiment, the medicament comprises a medicament for treating a spermatogenic disorder.
Compared with the prior art, the beneficial effects of the application include:
an embodiment of the application provides a construction method of a Ccdc182 gene knockout mouse model, which designs a sgRNA sequence of a target site and can efficiently and accurately construct the Ccdc182 gene knockout mouse model for subsequent functional research by using a CRISPR/Cas9 technology.
The CRISPR/Cas9 technology is used for constructing a Ccdc182 (NM_ 028859.1) gene knockout mouse animal model, and a good foundation is laid for further researching the function of the Ccdc182 gene and the genetic research of the Ccdc182 gene in the oligospermia of mice or humans.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort to a person skilled in the art.
FIG. 1 is a schematic diagram of a design strategy of a Ccdc182 gene knockout mouse model;
FIG. 2 is an identification electrophoretogram of a gene knockout mouse model;
FIG. 3 is a graph showing average litter size statistics in wild-type mice and Ccdc182 knockout mice models;
wherein cccc 182 wild type allele is a cccc 182 wild type allele; ccdc182 knockout allele is the Ccdc182 knockout allele; coding region is a coding region; the uncoding region is a non-coding region; mutation; primer localization is the primer position; pups per litter is the number of pups per litter; control is control; KO is knockdown.
Detailed Description
The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order to provide a more thorough understanding of the present disclosure. It is also to be understood that this application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but is capable of numerous changes or modifications without departing from the spirit of the application, as equivalent forms are intended to be within the scope of this application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application, it being understood that the present application may be practiced without one or more of these details.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Terminology
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Unless otherwise conflict with the purpose and/or technical solution of the present application, the present application relates to the cited documents which are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in this application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. Examples of the relevant technical features and preferred modes to be cited in the present application when the cited documents are referred to in the present application are incorporated by reference in the present application, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).
In this application, reference is made to a numerical interval (i.e., a numerical range), where the optional numerical distribution is considered continuous, and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints, unless otherwise indicated. Unless otherwise indicated, when a numerical range merely refers to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, herein, each integer is recited directly, such as t is an integer selected from 1-10, and t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.
Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of protection of the present application.
In this application, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the present application.
CRISPR/Cas9 technology: the gene is established based on the immune system reconstruction in bacteria and archaea, consists of an R-S structure formed by a plurality of regular short repeated sequences and non-repeated interval sequences and a gene operon for encoding Cas9 nuclease, and is characterized in that target DNA sequence recognition is carried out and DNA double strand break is caused by guiding RNA (sgRNA) mediated endonuclease Cas9 protein, and damaged DNA is restored in a homologous recombination or non-homologous end connection mode, so that multiple modifications such as fixed-point knockout, knock-in and gene correction of genes are realized on target sites. Because of the characteristics of high specificity, simple molecular construction and short flow, CRISPR/Cas9 technology has been rapidly developed in recent years.
Gene knockout using CRISPR/Cas9 technology requires two key factors, firstly the effective sgRNA guide sequence and then the presence of Cas9 protein. Compared with Zinc Finger Nuclease (ZFN) technology and transcription-like effector nuclease (TALEN) technology, the method has the advantages of being more and more widely applied due to the specificity, high efficiency, simplicity in design and the like of target editing target genes, has strong genome editing activity in bacteria, mammalian cells, zebra fish, mice, rats and the like, has the advantages of successfully constructing albinism mouse models by applying CRISPR/Cas9 gene knockout technology, has the advantages of obtaining liver function abnormal disease mouse models by knocking out PTEN genes through a tail vein high-pressure injection mode, helping people find potential treatment targets of related diseases, and has important roles in researching pathogenesis and drug screening of diseases.
The term "Ccdc182 gene" coded-coil domain-containing protein, ccdc182 (coded-coil domain containing) which is located on mouse chromosome 11, NC_000077.7 (88184869-88186087), 1 transcript in mouse (NM_ 028859.1), full length 1219bp, comprising 1 exon, the encoded protein comprising 153 amino acid residues.
The term "sgRNA" sgRNA (small guide RNA) is a guide RNA (gRNA) that directs insertion or deletion of uridine residues into the plastids (kinetoplastid) during RNA editing, a small non-coding RNA that can be paired with pre-mRNA.
The term "pseudopregnant female mouse" ligates male mice and female mice to mate so that the female mice are in a pseudopregnant state, and a reproductive physiological state similar to that of normal female mice is produced, and particularly, the female mice in the pseudopregnant state can normally secrete progesterone only by the corpus luteum formed after ovulation under mating stimulation, and the female mice in the pseudopregnant state are used as receptors for embryo transfer. Pseudopregnant female mice were subjected to oviduct transplantation, usually 0.5 days after the thrombus, and uterine transplantation, usually 2.5 days after the thrombus.
In one aspect, the application provides a kit for constructing a Ccdc182 gene knockout mouse model, wherein the kit comprises sgRNA1, sgRNA2 and corresponding primers.
The sequence of the sgRNA1 is shown as SEQ.ID.NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2; the sequence of the primer is shown as SEQ ID NO. 3-SEQ ID NO. 4.
gRNA1: GAAGGTGGCCGGGGTACAAAGGG（SEQ ID NO.1）
gRNA2: CCAACATGTTCTCCAACCGCTGG（SEQ ID NO.2）
Forward primer（F1）:5’-CTTTCAGGCAGGGTCCATTCT-3’（SEQ ID NO.3）
Reverse primer（R1）:5’-CGGAACTTCACCCGAAAACC-3’（SEQ ID NO.4）
In a specific example, the kit further comprises Cas9 mRNA. Cas9 mRNA can transiently express Cas9 protein in cells, and target DNA sequences are sheared under the guidance of crRNA and tracrRNA complexes, so that compared with a plasmid or virus system, the method effectively reduces off-target effect of CRISPR gene editing and is suitable for mammal cell gene editing.
Optionally, the kit further comprises one or more of a PCR reaction buffer and a PCR reaction solution.
Further alternatively, the PCR reaction solution comprises Taq DNA polymerase, dNTPs and Mg 2+ One or more of the following.
Taq DNA polymerase was the first thermostable DNA polymerase to be found, and has a molecular weight of 65kD, and was originally obtained by extraction from a strain of Thermophilus hydrophila (thermus aquaticus) isolated from hot springs by Saiki et al. The enzyme can resist high temperature, the residual activity of the enzyme is more than 90% of the original activity after the enzyme reacts for 2 hours at 70 ℃, the residual activity of the enzyme is 60% of the original activity after the enzyme reacts for 2 hours at 93 ℃, and the residual activity of the enzyme is 40% of the original activity after the enzyme reacts for 2 hours at 95 ℃.
Taq DNA polymerase can be used for DNA sequencing in molecular cloning and specific fragments of DNA can be amplified in vitro using polymerase chain reaction (polymerase chain reaction, PCR). During the PCR process, since Taq DNA polymerase is not inactivated in the denaturation step (about 94 ℃) and can directly enter the second cycle, new enzyme is not needed to be added in each cycle, which makes Taq DNA polymerase the unique enzyme in the PCR reaction.
dNTPs or deoxyribonucleotides are nucleotides that contain deoxyribose as a sugar molecule. These nucleotides are referred to as DNA nucleotides. In addition, there are four dntps; they are dATP, dGTP, dGTP and dTTP. dNTPs are, from their function, precursor molecules of DNA. The main function of DNA nucleotides is to transfer genetic information from one generation to the next by DNA.
In addition, three components of dNTPs are deoxyribose, a nitrogenous base, and a phosphate group. Deoxyribose contains a 3' OH group. Nitrogenous bases include purine bases or pyrimidine bases. In addition, the nitrogenous bases form hydrogen bonds to form base pairs during DNA synthesis. During DNA synthesis, the 3'oh group forms a phosphodiester bond with the phosphate group in the 4' carbon of the second deoxyribonucleotide.
In another aspect, the present application further provides a method for constructing a Ccdc182 gene knockout mouse model, which includes the following steps:
(1) The sgRNA1 and the sgRNA2 targeted to the mouse Ccdc182 gene were obtained.
The sequence of the sgRNA1 is shown as SEQ.ID.NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2.
(2) And constructing an sgRNA/Cas9 expression vector by adopting the sgRNA1 and the sgRNA2, and performing in vitro transcription to prepare mRNA.
(3) The mRNA was introduced into pseudopregnant female mice to prepare F0 mice.
(4) And (3) screening a mice genetic line with stable Ccdc182 gene knockout from the F0 generation mice, hybridizing a positive F0 generation mouse with a wild type mouse to obtain an F1 generation heterozygote mouse, and mating the F1 generation heterozygote mouse to obtain a F2 generation Ccdc182 gene knockout homozygous mouse with stable genetic variation.
In a specific example, the mouse strain is selected from C57BL/6. C57BL/6, often referred to as "C57 black 6", "C57" or "black 6" (standard abbreviated as B6), is a common inbred strain of laboratory mice. The gene can be widely used as a transgenic mouse in genetic experiments to simulate human gene defect diseases, and has the characteristics of being applicable to congeneric lines, easy to reproduce, strong in physique and the like.
Alternatively, positive mice are identified by PCR, and primers used in the screening by PCR are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Further alternatively, the amplification procedure when screening by PCR method is: reacting for 4-6 min at 94-96 ℃; reacting for 25-35 s at 93-95 ℃; reacting for 28-32 s at 55-65 ℃; reacting for 38 s-42 s at 70-74 ℃ for 35 cycles; reacting for 4-6 min at 70-74 ℃ and preserving at 3.5-4.5 ℃.
For example, 94 ℃, 95 ℃ and 96 ℃ for 4min, 5min and 6min; the reaction is carried out at 93 ℃, 94 ℃, 95 ℃ for 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34s, 35s; conditions of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ for 4min, 5min, 6min; the reaction is carried out at 93 ℃, 94 ℃, 95 ℃ for 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34s, 35s; conditions of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ for 28s, 29s, 30s, 31s, 32s; the conditions of 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ and 38s, 39s, 40s, 41s and 42s are reacted for 35 cycles; the reaction was carried out at 70℃and 71℃and 72℃and at 73℃and 74℃for 4min, 5min and 6min, and the reaction was carried out at 3.5℃and 4℃and 4.5℃for storage.
The application also provides application of the kit or the Ccdc182 gene knockout mouse model obtained by the construction method in drug screening, wherein the drugs comprise drugs for treating spermatogenic disorders.
Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are preferably referred to in the guidelines given in the present application, may be according to the experimental manual or conventional conditions in the art, may be according to the conditions suggested by the manufacturer, or may be referred to experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
Example 1
1. Construction of Ccdc182 Gene knockout mouse model
(1) For the loss-of-function mutation at cccc 182 (nm_ 028859.1), a corresponding targeting target site sgRNA sequence was designed.
(2) Cas9 mRNA and gRNA are obtained by means of in vitro transcription; cas9 mRNA and gRNA were microinjected into fertilized eggs of C57BL/6J mice to obtain F0 mice. PCR amplification and sequencing identification positive F0 generation mice are mated with C57BL/6J mice to obtain 2 positive F1 generation mice, and the design strategy is shown in figure 1.
(3) Genotyping of F0 mice: the fertilized eggs after injection are transplanted into pseudopregnant female mice, and the mice born for about 20 days are F0 generation mice. Genotyping was performed by PCR amplification and sequencing. Because the early cleavage speed of fertilized eggs is very fast, the obtained F0 generation mice are chimeric and do not have the capability of stable inheritance, and the F1 generation mice which can be inherited stably need to be passaged.
2. Genotyping of mice
2.1 extraction of DNA from mouse tissue samples, using the gDNA extraction kit from Tiangen (TIANamp Genomic DNA Kit, DP 304-03).
The method comprises the following steps:
(1) The toe or tail of the mice was added with 200 μl of buffer GA and the tissues were minced using alcohol sterilized scissors.
(2) Add 20. Mu.L of Proteinase K solution and mix upside down.
(3) The EP tube was inserted into the float and placed in a 56 ℃ water bath for 1h with 1h of mixing upside down.
(4) 200. Mu.L of buffer GB was added and mixed well upside down and left at 70℃for 10min.
(5) 200 mu L of absolute ethyl alcohol is added, and after being mixed uniformly upside down, the mixture is vigorously vibrated on a vibrator for 15sec.
(6) Centrifuging the mixed solution in the adsorption column which is placed in a collecting pipe and marked, carrying out 12000rcf/min for 30sec, discarding the collecting pipe, and placing the adsorption column in a brand new collecting pipe.
(7) 500. Mu.L of buffer GD was added to the column, mixed upside down, centrifuged at 12000rcf/min for 30sec, the collection tube was discarded, and the column was placed in a fresh collection tube.
(8) 600. Mu.L of the rinse PW was added to the column, mixed upside down, centrifuged at 12000rcf/min for 30sec, the collection tube was discarded, and the column was placed in a fresh collection tube.
(9) And (8) repeating the step 8.
(10) The column was allowed to air-stand at 12000rcf/min for 2min, the collection tube was discarded and the column was placed in a fresh 1.5mL EP tube and allowed to air-stand for 5min.
(11) Suspending and dripping 250 mu L TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging for 1 min at 12000rcf/min, removing the adsorption column, covering the EP tube, and marking.
After the gDNA extraction is completed, PCR amplification and sequencing are performed using the primers F1/R1.
The PCR reaction system is as follows: 10 mu L Green mix Buffer, 1 mu L gDNA, 1 mu L F1, 1 mu L R1, finally with ddH 2 O was made up to 20. Mu.L.
The PCR reaction procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 60 ℃ for 60s; extending at 72 ℃ for 60s, and 34 cycles in total; extension at 72℃for 5min and finally the procedure was ended at 4 ℃. After the procedure is completed, the PCR amplified products are subjected to agarose gel electrophoresis to determine the band size.
The genotyping primer sequences used therein are as follows:
Forward primer （F1）: 5’-CTTTCAGGCAGGGTCCATTCT-3’（SEQ ID NO.3）
Reverse primer （R1）: 5’-CGGAACTTCACCCGAAAACC-3’（SEQ ID NO.4）
2.2 Mouse gDNA PCR
The products of the PCR amplification of the mice with primers F1/R1 were electrophoresed on a 2% agarose gel, see the electrophoretogram shown in FIG. 2:
wild type: the P1 and P2 PCR gave a single 625bp fragment.
Heterozygotes: p1 and P2 PCR gave two fragments of 625bp and 373 bp.
Homozygote: the P1 and P2 PCR gave a single 373bp fragment.
3. Model identification:
the mice of different genotypes were further identified for fertility, and the lower panel is the average litter size statistics for wild-type mice and Ccdc182 knockout model mice. In the figure, black circles represent control males and black squares represent homozygous males. Statistical analysis was performed between the different groups using T-test, which indicates that p.ltoreq.0.05 between the two groups, and the results are shown in fig. 3.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing teachings, and equivalents thereof will be within the scope of the present application. It should also be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, which description and drawings may be interpreted accordingly.
Claims (10)
1. A kit for constructing a Ccdc182 gene knockout mouse model, which is characterized by comprising sgRNA1 and sgRNA2;
the sequence of the sgRNA1 is shown as SEQ.ID.NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2.
2. The kit for constructing a Ccdc182 gene knockout mouse model according to claim 1, further comprising a corresponding detection primer;
the sequence of the detection primer is shown as SEQ ID NO. 3-SEQ ID NO. 4.
3. The kit for constructing a Ccdc182 gene knockout mouse model according to claim 1, wherein the kit further comprises Cas9 mRNA.
4. The kit for constructing a Ccdc182 gene knockout mouse model according to claim 1, wherein the kit further comprises Taq DNA polymerase, dNTPs and Mg 2+ One or more of the following.
5. The construction method of the Ccdc182 gene knockout mouse model is characterized by comprising the following steps:
(1) Obtaining sgRNA1 and sgRNA2 of a targeted mouse Ccdc182 gene;
the sequence of the sgRNA1 is shown as SEQ.ID.NO. 1; the sequence of the sgRNA2 is shown as SEQ ID NO. 2;
(2) Constructing an sgRNA/Cas9 expression vector by adopting the sgRNA1 and the sgRNA2, and performing in vitro transcription to prepare mRNA;
(3) Introducing the mRNA into a pseudopregnant female mouse body to prepare an F0 generation mouse;
(4) And (3) screening a mice genetic line with stable Ccdc182 gene knockout from the F0 generation mice, hybridizing a positive F0 generation mouse with a wild type mouse to obtain an F1 generation heterozygote mouse, and mating the F1 generation heterozygote mouse to obtain an F2 generation Ccdc182 gene knockout homozygous mouse with stable genetic variation.
6. The method of claim 5, wherein the strain of mice is selected from the group consisting of C57BL/6.
7. The method for constructing a model of a Ccdc182 gene knockout mouse according to claim 5 or 6, wherein a PCR method is used to identify positive mice, and the detection primers used in the screening by the PCR method are shown as SEQ ID NO.3 and SEQ ID NO. 4.
8. The method for constructing a Ccdc182 gene knockout mouse model according to claim 5 or 6, wherein the amplification procedure when screening by PCR method is: reacting for 4-6 min at 94-96 ℃; reacting for 25-35 s at 93-95 ℃; reacting for 28-32 s at 55-65 ℃; reacting for 38 s-42 s at 70-74 ℃ for 35 cycles; reacting for 4-6 min at 70-74 ℃ and preserving at 3.5-4.5 ℃.
9. Use of the kit according to any one of claims 1 to 4 or the Ccdc182 gene knockout mouse model obtained by the construction method according to any one of claims 5 to 9 in drug screening.
10. Use according to claim 9, characterized in that the medicament comprises a medicament for the treatment of spermatogenic disorders.
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