Construction method and application of cre tool mouse for gene specific knockout of germ cells

Abstract

The invention relates to the technical field of biological medicines, in particular to a construction method and application of a tool mouse for specifically knocking out cre by germ cell genes, and the construction method is characterized in that a Cre recombinant sequence carrying a Ddx4 promoter sequence is knocked into a mouse genome safety site Hipp11 by virtue of a CRISPR-CAS9 technology so as to construct a Ddx4 Promoter-Kozak-CreERT2-rBGpA gene knock-in mouse model. The model mouse realizes the characteristic of specific expression of CreERT2 recombinase under the driving of a germ cell specific promoter Ddx4, the expressed CreERT2 protein is inactivated after the cell cytoplasm is combined with the HSP90 protein, the CreERT2 is dissociated after being induced by tamoxifen and enters a cell nucleus, and an Loxp sequence is cut to realize gene recombination. According to the invention, the exogenous sequence is inserted into the safe site of the genome, so that the expression and function of the original endogenous gene of the mouse are not influenced, the fertility of the male and female mice is not influenced, and the method has the advantages of high efficiency and convenience in subsequent gene knockout. The model mouse is expected to accelerate the analysis of the action mechanism for regulating the gametogenesis gene of the mouse.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a construction method of a germline cell gene-specific knockout Cre tool mouse and application thereof. BACKGROUND

[0002] Infertility is an important reproductive health problem that plagues couples of childbearing age in today's society. Survey data shows that about 15% of couples seek medical help due to infertility. There are many factors that cause infertility, and genetic factors caused by gene function disorders are undoubtedly the most important reason, which often induces abnormal gametes or gamete dysfunction. Therefore, exploring new genes or new functions of known genes that regulate the reproductive process, and analyzing the function and mechanism of important genes affecting infertility have important practical needs and clinical value for the diagnosis and treatment of infertility diseases.

[0003] All physiological activities including the reproductive process cannot be carried out normally without the precise and orderly regulation of genes. Therefore, analyzing the function of genes is of great significance to understand the physiological function regulation mechanism of the body. The common means of analyzing gene function is to use gene knockout or overexpression strategies in specific cells or model animals. However, a large number of genes are involved in the function regulation of multiple tissues and organs, which leads to the fact that when using conventional whole-body gene knockout models to study the function of a specific gene in a specific cell, the influence of other cells cannot be ruled out. In addition, some very important genes cause embryonic lethality, premature birth or death due to whole-body knockout. For the above reasons, using tissue-specific knockout strategies to study the function of the gene of interest in a specific cell is undoubtedly the best choice, and the most commonly used is the "Cre-Loxp" system. To achieve specific knockout of genes in germ cells using this system, a tool mouse is needed that expresses Cre enzyme driven by a germ cell-specific gene promoter.

[0004] There are many Cre tool mice that can achieve constitutive specific knockout of germ cells, such as Ddx4-Cre, Ngn3-Cre, PrP-CreERT, Stra8-Cre, Spo11-Cre, Wisp3-Cre, Sycp1-Cre, Prm1-Cre, Acrv1-Cre, etc. These tool mice can achieve knockout of target genes in specific germ cells such as spermatogonia or spermatocytes. Subsequent researchers have developed tool mice that start to initiate knockout after tamoxifen induction, such as FVB / N-Tg(Ddx4-cre / ERT2)1Dcas / J, B6-Ddx4 em1(CreERT2)Utr to knock out the target gene at any stage of specific germ cells. However, these tool mice all have different degrees of defects. The first tamoxifen-induced tool mouse is constructed using traditional transgenic technology, and the insertion site is unknown, the genetic rarity is rare, and the specificity of gene recombination is not fully evaluated. In order to overcome the above defects, the second tool mouse uses CRISPR-CAS9 technology to insert CreERT2 before the stop codon of Ddx4 gene, in order to realize the purpose of allowing the endogenous Ddx4 promoter to drive the expression of CreERT2 gene without affecting the expression of Ddx4 gene. However, unfortunately, the mouse obtained by this construction strategy finally affects the function of Ddx4 protein, and the transcript level of homozygous mouse is significantly reduced, which finally leads to the sterility of homozygous male mice due to the arrest of germ cell development at the round spermatid stage, which greatly limits the convenience of using the mouse. Only heterozygous mice can be used to mate with Flox mice, and the efficiency of conditional gene knockout is greatly reduced.

[0005] To realize the expression of genes driven by specific promoters, in addition to using the above in situ knockout method, insertion at safe sites in the genome such as Col1a1, Rosa26, H11, etc. is also a common strategy. The safe site H11 (also known as Hipp11) is a verified safe harbor locus in the mouse genome, which was first discovered and named by Simon Hippenmeyer in 2010. It is located in the intergenic region of mouse chromosome 11, specifically between the Eif4enif1 gene (19th exon) and the Drg1 gene (9th exon), with a length of about 5 kb. Its advantages are that the insertion of exogenous genes does not affect the function of endogenous genes, the flanking sequence has a wide spatial and temporal EST (expressed sequence tag) expression pattern, which can drive the stable expression of exogenous genes in multiple tissues and developmental stages, homozygous knock-in mice develop normally and are fertile, have no abnormal phenotypes, have small expression differences among cells in the same tissue, have high genetic stability, etc.

[0006] Therefore, the present application uses CRISPR-CAS9 technology to knock in the Cre recombination sequence carrying the Ddx4 promoter sequence into the mouse genome safe site Hipp11 to construct a "Ddx4 Promoter-Kozak-CreERT2-rBGpA" gene knock-in mouse model (C57BL / 6J-Igs2 em1(Ddx4-Kozak-CreERT2-rBGpA) NTUXi, B6-Igs2 (Ddx4CreERT2)The model mouse realizes the characteristics of specific expression of CreERT2 recombinase under the driving of the germ cell-specific promoter Ddx4, and when induced by tamoxifen, the CreERT2 dissociates into the nucleus to play a recombinase activity, and cuts the sequence of two Loxp sites to realize gene recombination. The mouse obtained by the application does not affect the expression and function of the endogenous genes of the original mouse, and does not affect the fertility of male and female mice, and can be used for breeding and realizing gene knockout of homozygous mice, and is more convenient and efficient. It is expected to accelerate the analysis of the mechanism of regulating mouse gametogenesis genes, explore new genes affecting fertility or new functions of known genes in germ cells, and provide candidate target genes and intervention strategies for diagnosis and treatment of human infertility diseases. SUMMARY

[0007] The purpose of the present application is to solve the problems existing in the prior art, and a construction method of a germ cell gene-specific knockout cre tool mouse and its application are proposed. The model mouse is expected to accelerate the analysis of the mechanism of regulating mouse gametogenesis genes, explore new genes affecting fertility or new functions of known genes in germ cells, and provide candidate target genes and intervention strategies for diagnosis and treatment of human infertility diseases.

[0008] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0009] A construction method of a germ cell gene-specific knockout cre tool mouse, a Ddx4 Promoter-Kozak-CreERT2-rBGpA sequence inducing gene recombination guide RNA (gRNA) is inserted into a H11 safe site. The gRNA is guided to be inserted downstream of the 19th exon of Eif4enif1.

[0010] Preferably, the sequence of the Ddx4 Promoter-Kozak-CreERT2-rBGpA sequence inducing gene recombination guide RNA (gRNA) is: GAACACTAGTGCACTTATCCTGG.

[0011] The present application also provides a cre tool mouse constructed according to the above-mentioned method for detecting B6-Igs2 (Ddx4CreERT2) The application of the primer combination for distinguishing whether the NTUXi gene knockout mouse is successfully constructed and whether the mouse genotype is heterozygous or homozygous, and the specific primer sequences are as follows: the PCR product of the combination of F3 and R1 can identify the knockout sequence, and the product length is 386 bp; the PCR product of the combination of F3 and R3 can confirm that the site is a wild type sequence, and the product length is 519 bp; the sequences of the three specific primers are as follows:

[0012] R1: 5'-ACTCCCAGTTCATGGCAAATAGAA-3';

[0013] F3: 5'-CTCTACTGGAGGAGGACAAACTG-3';

[0014] R3: 5'-GTCTTCCACCTTTCTTCAGTTAGC-3'.

[0015] In the present application, the tool mouse is officially named C57BL / 6J-Igs2 according to the naming specification of genetically engineered mice. em1 (Ddx4-Kozak-CreERT2-rBGpA) NTUXi, referred to as B6-Igs2 (Ddx4CreERT2) NTUXi.

[0016] By adopting the above technical scheme: the tool mouse for realizing induced specific knockout of genes in germ cells is invented by means of CRISPR-CAS9 technology, and the timed and site-specific knockout of target genes is realized through mating with Flox mice and administration of tamoxifen.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application obtains a new tool mouse B6-Igs2 by designing a set of gRNA and identification primer combination for gene knock-in mice. (Ddx4CreERT2) NTUXi, the model mouse has the following significant advantages compared with the existing tool mice of the same type:

[0019] 1. The designed gRNA guided recombination does not affect the function of the endogenous DDX4 protein of the tool mouse, reducing its impact on the research of germ cell function;

[0020] 2. The female and male homozygous individuals of the mouse do not affect their own gametogenesis and function, and have normal fertility;

[0021] 3. The mouse can be bred by homozygous mating and the target gene loxp site can be cleaved and recombined, which greatly improves the efficiency and is more convenient to use. Based on this, the use of the tool mouse provided by the present application is expected to accelerate the research and development of the diagnosis and treatment of infertility diseases. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 To construct B6-Igs2 (Ddx4CreERT2) NTUXi gene editing mouse and genotype identification strategy diagram;

[0023] Figure 2 Electrophoresis result map for identifying the genotype of offspring of the gene edited mice obtained in Example 1 of the present application using R1, R3 and F3, Het (heterozygote), Hom (homozygote), WT (wild type);

[0024] Figure 3 Fertility test result map of the homozygous gene edited mice obtained in Example 1 of the present application; A is B6-Igs2 (Ddx4CreERT2) Fertility test result of NTUXi homozygous male mice; B is B6-Igs2 (Ddx4CreERT2) Fertility test result of NTUXi homozygous female mice;

[0025] Figure 4 B6-Igs2 verification result map using Rosa-LSL-tdtomato reporter mice in Example 2 of the present application; (Ddx4CreERT2) NTUXi tool mouse gene recombination efficiency and specificity result map; A is the result map of tdTomato reporter mice showing gene recombination only in testes after tamoxifen induction; B is the control mouse without induction, no gene recombination occurred in each tissue. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings, so that the persons skilled in the art can better understand the advantages and features of the present application, and the protection scope of the present application can be defined more clearly. The described embodiments of the present application are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the persons skilled in the art without creative labor fall within the protection scope of the present application.

[0027] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0028] The materials, reagents and the like used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] Example 1

[0030] Construction of B6-Igs2 using CRISPR-CAS9 technology (Ddx4CreERT2) NTUXi gene knock-in mouse.

[0031] 1. The designed gRNA sequence is entrusted to the synthesis of the company, and the BCA cloning is used as a template to construct the knock-in sequence Ddx4 Promoter-Kozak-CreERT2-rBGpA with a homologous arm, and the knock-in vector is obtained. The gRNA insertion site, the site of the targeted insertion sequence, and the primer design site for identifying the success of insertion designed by the application are shown in Figure 1 .

[0032] 2. Then the above-mentioned gRNA, the vector with the knock-in sequence and the CAS9 mRNA purchased from a commercial company are microinjected into the fertilized eggs of C57BL / 6J mice.

[0033] 3. The fertilized eggs are transplanted into surrogate mother mice, and then the insertion region is amplified by designing primers to identify the successful F0 generation of positive mice by Sanger sequencing.

[0034] 4. The F0 generation mice are mated with WT mice to produce F1 generation mice, which are identified by conventional genotyping Figure 2 ) and Northern Blot to obtain the heterozygous F1 generation mice.

[0035] 5. The F1 generation heterozygous mice are self-crossed to produce homozygous offspring for growth and development evaluation and fertility test, as shown in Figure 3 , the homozygous B6-Igs2 (Ddx4CreERT2) NTUXi female and male mice do not affect their own fertility.

[0036] Example 2

[0037] B6-Igs2 (Ddx4CreERT2) Verification of the recombination efficiency and specificity of the LoxP site induced by the NTUXi tool mouse.

[0038] 1. Rosa-LSL-tdtomato (abbreviated as tdtomato) reporter gene mice were purchased from Saiye Biotechnology Co., Ltd. to evaluate the gene knockout efficiency of the tool mouse constructed by the application. This mouse is a widely used genetically engineered mouse model in biomedical research, and its core function is as a reporter system for Cre recombinase. This system drives the expression of tdTomato (a red fluorescent protein) through the CAG promoter, but its expression is regulated by the loxP-flanked STOP cassette. In the absence of Cre recombinase, the STOP cassette prevents the transcription of tdTomato, thereby avoiding the expression of the fluorescent protein. When Cre recombinase is expressed in specific tissues or cells, it mediates recombination between loxP sites, thereby excising the STOP cassette, allowing the CAG promoter-driven tdTomato to be expressed, producing a red fluorescent signal.

[0039] 2. tdtomato mice and B6-Igs2 (Ddx4CreERT2) NTUXi mating generates double heterozygous mice carrying both genes.

[0040] 3. The double heterozygous mice were intraperitoneally injected with tamoxifen (2 mg / 30 g body weight) for 5 consecutive days to induce the Cre enzyme to exert its recombination effect.

[0041] 4. Two days after the end of drug administration, the mice were dissected and the expression intensity of red fluorescent protein in various tissues of the mice was observed under a living imaging system. Figure 4 As shown in the figure, high-intensity red fluorescence signals were detected only in the testis, indicating that the B6-Igs2 constructed by the present invention (Ddx4CreERT2) NTUXi tool mice can achieve specific inducible knockout of genes in testicular germ cells.

[0042] 5. Since the Ddx4 gene is expressed not only in male germ cells but also in female germ cells, although this method was not used in the present invention to evaluate its knockout effect on female germ cells, it should theoretically be possible to experiment with specific knockout of germ cells in female mice.

[0043] In summary, the present invention designs a set of methods for constructing B6-Igs2 (Ddx4CreERT2) The gRNA sequence of the NTUXi Cre tool mouse and its genotype identification primer combination. The tool mouse constructed using this invention does not affect the expression of endogenous genes or their own fertility, and can achieve the purpose of efficient, specific, and inducible knockout of germ cell genes.

[0044] The descriptions and practices disclosed in this invention are easy to understand and comprehend for those skilled in the art, and modifications and refinements may be made without departing from the principles of the invention. Therefore, modifications and improvements made without departing from the spirit of the invention should also be considered within the scope of protection of this invention.

Claims

1. A method for constructing a germline cell gene-specific knockout cre tool mouse, characterized by, The gRNA for inducing gene recombination of the Ddx4 Promoter-Kozak-CreERT2-rBGpA sequence inserted in the H11 safe site.

2. The method according to claim 1, wherein the method is characterized by, The sequence of the gRNA for inducing gene recombination of the Ddx4 Promoter-Kozak-CreERT2-rBGpA sequence is: GAACACTAGTGCACTTATCCTGG.

3. A cre tool mouse constructed according to the method of any one of claims 1-2 for detecting B6-Igs2 (Ddx4CreERT2) application of a primer combination for determining whether the NTUXi gene knock-in mouse is constructed successfully and distinguishing whether the mouse genotype is heterozygous or homozygous, characterized in that, The specific primer sequences are as follows: the PCR product of the combination of F3 and R1 is used for identifying the knock-in sequence, and the product length is 386 bp; the PCR product of the combination of F3 and R3 is used for confirming that the site is a wild type sequence, and the product length is 519 bp; the sequences of the three specific primers are as follows: R1: 5'-ACTCCCAGTTCATGGCAAATAGAA-3'; F3: 5'-CTCTACTGGAGGAGGACAAACTG-3'; R3: 5'-GTCTTCCACCTTTCTTCAGTTAGC-3'.

Images