A method for constructing a tool mouse model expressing cre specifically in liver stem cells

Abstract

The application discloses a method for constructing a tool mouse model for specifically expressing Cre in liver stem cells, and relates to the technical field of genetic engineering. The method comprises the following steps: after female mice are mated with male mice, fertilized eggs in a single cell stage are collected, and then a transgenic vector, sgRNA and Cas9 protein are microinjected into a male pronucleus; the fertilized eggs which develop into two cells are transplanted into the uterus of a pseudopregnant female mouse, and a transgenic F0 generation mouse is obtained through foster breeding; a transgenic founder mouse is screened out; the transgenic founder mouse is mated with a wild-type mouse, and a line is established through subculture to obtain an F1 generation mouse; the F1 generation mouse is subjected to genotype identification, and the tool mouse model is obtained. The tool mouse model constructed by the application can be applied to inducing LoxP recombination in liver stem cells, so as to conditionally knock out a target gene in liver stem cells, and the function of the target gene can be researched or a disease model can be constructed.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for constructing a tool mouse model that specifically expresses Cre in liver stem cells. Background Technology

[0002] Gene knock-in is a technique that utilizes homologous recombination to introduce a foreign functional gene (a gene that was not originally present in the genome or has been inactivated) into a cell. This gene then undergoes homologous recombination with a homologous sequence in the genome, inserting itself into the genome and achieving expression within the cell. The type II CRISPR / Cas9 system, developed in recent years, has become an important tool for transgenic technology. The type II CRISPR / Cas9 system consists of a single guide RNA (sgRNA) and CRISPR-associated protein 9 (Cas9). The Cas9 protein, mediated by the sgRNA, recognizes and cleaves DNA sequences complementary to the sgRNA, causing double-strand breaks (DSBs). DSBs can be repaired through homologous end joining (HEJ), thereby enabling the introduction of specific genes. Due to its simplicity, low cost, and ability to cut large fragments of target genes, type II CRISPR / Cas9 technology is a promising molecular tool for site-specific genome editing.

[0003] Cre recombinase was discovered in 1981 from P1 bacteriophage and belongs to the λInt enzyme supergene family. The full-length coding sequence of the Cre recombinase gene is 1029 bp, encoding a 38 kDa monomeric protein composed of 343 amino acids. It not only possesses catalytic activity but also, similar to restriction enzymes, recognizes specific DNA sequences, namely LoxP sites, and mediates specific recombination between two LoxP sites (sequences), resulting in deletion or recombination of the gene sequence between LoxP sites. Cre recombinase has a recombination efficiency of 70%, operates without any cofactors, and can act on DNA substrates of various structures, such as linear, circular, and even supercoiled DNA.

[0004] Conditional knockout mice are widely used experimental models that play an important role in studying the relationship between genes and the occurrence and development of diseases. By using the chromosome site-specific recombinase system Cre-LoxP, recombinant heterozygous mice can be crossed with Cre transgenic mice at the individual level, and the offspring mice can be screened to obtain conditional knockout mice with the deletion of exogenous marker genes.

[0005] Existing Cre mice have limitations: currently, liver stem cells lack specific biomarkers, therefore, there is a lack of tool mice that specifically express Cre recombinase in liver stem cells. Therefore, there is an urgent need to establish a tool mouse model that specifically expresses Cre in liver stem cells. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a tool mouse model that specifically expresses Cre in liver stem cells, in order to solve the problems existing in the prior art. The tool mouse model constructed by this invention is a mouse model that specifically expresses Cre through Ezh2, which can be used to induce LoxP recombination in liver stem cells, thereby conditionally knocking out the target gene in liver stem cells, and studying the function of the target gene or constructing a disease model.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides an Ezh2-P2A-iCre transgenic vector, comprising an Ezh2-P2A-iCre promoter fragment, the nucleotide sequence of which is shown in SEQ ID NO.7.

[0009] The present invention also provides the application of the above-described transgenic vector in the preparation of a tool mouse model that specifically expresses Cre in liver stem cells.

[0010] This invention also provides a method for constructing a tool mouse model that specifically expresses Cre in liver stem cells, comprising the following steps:

[0011] (1) After mating between male and female mice, fertilized eggs in the single-cell stage were collected. Then, the male pronuclear microinjection vector, sgRNA and cas9 protein were injected. The fertilized eggs that developed normally into two cells were transplanted into the uterus of a pseudopregnant female mouse to obtain transgenic F0 generation mice. The F0 generation mice were identified and the founder mice of Ezh2-P2A-iCre transgene were screened.

[0012] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3;

[0013] (2) The founder mice of Ezh2-P2A-iCre transgenic were crossed with wild-type mice, and the F1 generation mice were obtained by passage and genotyping of the F1 generation mice to obtain the tool mouse model.

[0014] Further, in step (1), the method for preparing the sgRNA includes:

[0015] a) Using pUC57kan-T7 plasmid as a template, the sgRNA coding region was amplified by PCR using the primer pair shown in SEQ ID NO.1-2. The sgRNA coding region and the pUC57kan-T7 vector were then ligated to obtain the sgRNA-pUC57kan-T7 plasmid.

[0016] b) The sgRNA-pUC57kan-T7 plasmid was transcribed and purified in vitro to obtain the sgRNA.

[0017] The present invention discloses the following technical effects:

[0018] The Ezh2 gene is expressed in adult stem cells of various tissues (including gonadogenesis, neurogenesis, myogenesis, osteogenics, hematopoiesis, lymphogenesis, adipogenesis, epidermal differentiation, and hepatogenesis), maintaining stem cell stemness and influencing cell fate. The transgenic mouse model Ezh2-P2A-iCre constructed in this invention is a mouse specifically expressing Cre from Ezh2. It can be used to induce LoxP recombination in tissue stem cells, thereby conditionally knocking out the target gene in tissue stem cells, and to study the function of the target gene or construct disease models. When Ezh2-P2A-iCre mice are crossed with mice carrying the fluxed gene, this mouse model can be used for conditional knockout of genes in liver stem cells and for stem cell maintenance and cell lineage determination (including gonadogenesis, neurogenesis, myogenesis, osteogenics, hematopoiesis, lymphogenesis, adipogenesis, epidermal differentiation, and hepatogenesis).

[0019] This invention inserts Cre into the 3'UTR region of the Ezh2 gene instead of an exon, thus producing homozygous mice that express Cre while the Ezh2 gene remains intact. Furthermore, an advantage of this invention is that it avoids the insertion of the resistance gene Neo, thus eliminating the need for mating with Fip mice to remove Neo. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The structural diagram of the pUC57kan-T7-delG plasmid constructed in Example 1;

[0022] Figure 2 The structural diagram of the transgenic plasmid Ezh2-P2A-iCre constructed in Example 2;

[0023] Figure 3 Functional diagram of the Ezh2-P2A-iCre transgenic mouse constructed in Example 3;

[0024] Figure 4 This is a diagram of the identification strategy for F1 generation mice in Example 3; Wild type: ①② PCR reaction did not obtain the Target band, ③ PCR reaction can obtain a single WT band; Heterozygote: ①② PCR reaction can obtain the Target band, ③ PCR reaction can obtain a single WT band; Homozygote: ①② PCR reaction can obtain the Target band, ③ PCR reaction did not obtain a single WT band.

[0025] Figure 5 This is a diagram showing the identification results of the F1 generation in Example 3; where the numbers represent the rat tail number, WT represents the wild type C57BL / 6JGpt, N represents the negative blank control, and M represents the DNA Marker.

[0026] Figure 6 The results of Ezh2 expression detection in the liver in Example 3;

[0027] Figure 7 The results show the expression of Ezh2-P2A-iCre mice in the whole body in Example 3. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] In the following examples, the pUC57kan-T7 vector and PMD-18T plasmid were purchased from Takara Bio Inc.

[0034] Example 1: Construction of sgRNA

[0035] Forward and reverse primers targeting the sgRNA coding region were designed, and BsaI restriction sites were added to the primers. Using mouse genomic DNA as a template, the sgRNA coding region was amplified by PCR. The sgRNA coding region and the pUC57kan-T7 vector were digested with BsaI and ligated to obtain the pUC57kan-T7-delG plasmid. The pUC57kan-T7-delG plasmid was transcribed in vitro using the HiScribe T7 Quick Yield RNA Synthesis Kit and purified to obtain the sgRNA.

[0036] Primers for PCR amplification of the sgRNA coding region:

[0037] Forward primer (SEQ ID NO.1): GGTCTCTGTGGGCATCGAACGAGAAATGG.

[0038] Reverse primer (SEQ ID NO.2): GGTCTCCCATTTCTCGTTCGATGCCCACA.

[0039] sgRNA (SEQ ID NO. 3): TGTGGGCATCGAACGAGAAATGG.

[0040] The structure of the pUC57kan-T7-delG plasmid is as follows: Figure 1 Show.

[0041] Example 2 Construction of the transgenic vector expressing Ezh2-P2A-iCre

[0042] A. PCR amplification of 5'arm (SEQ ID NO.4), 3'arm (SEQ ID NO.5) and P2A-iCre (SEQ ID NO.6), followed by electrophoresis gel extraction and recovery for later use;

[0043] Using mouse genomic DNA as a template, the 5'arm fragment was amplified by PCR using primers with the upstream and downstream sequences of the 5'arm fragment (Table 1); the 3'arm fragment was obtained by PCR using primers with the upstream and downstream sequences of the 3'arm fragment (Table 2); and the P2A-iCre fragment was obtained by PCR using pAAV.cTNT.iCre plasmid as a template and primers with the upstream and downstream sequences of the P2A-iCre fragment (Table 3). The fragments were recovered from the electrophoresis gel for later use. The PMD-18T tool vector was linearized using NotI+SpeI, and the 5'arm, 3'arm, and P2A-iCre fragments were ligated to the enzyme-digested PMD-18T vector using SLIC to obtain the Ezh2-P2A-iCre fragment (SEQ ID NO.7).

[0044] Table 1

[0045]

[0046] Note: In the Forward primer, the underline represents the homologous region with the PMD-18T vector; the italic represents the EZH23'UTR homologous region; in the Reverse primer, the underline represents the iCre overlapping fragment; the italic represents the EZH23'UTR homologous fragment.

[0047] Table 2

[0048]

[0049] Note: In the Forward primer, italics represent iCre overlapping fragments; underline represents EZH23'UTR homologous fragments; in the Reverse primer, underline represents the homologous region with the PMD-18T vector; italics represent iCre overlapping fragments.

[0050] Table 3

[0051]

[0052] The connection system is shown in Table 4:

[0053] Table 4

[0054]

[0055]

[0056] B. Incubate the above ligation system at 50°C for 15 minutes. After incubation, store the sample on ice for subsequent transformation to obtain the transgenic plasmid Ezh2-P2A-iCre, with the structure shown below. Figure 2 As shown in the figure. The recombinant vector was transformed into DH5α Escherichia coli and plated with ampicillin-resistant plates; the resulting vector was then digested with enzymes and sequenced for verification.

[0057] SEQ ID NO.4:

[0058]

[0059] SEQ ID NO.5:

[0060]

[0061] SEQ ID NO.6:

[0062]

[0063] SEQ ID NO.7:

[0064]

[0065] Example 3: Construction of the Ezh2-P2A-iCre transgenic mouse model

[0066] 1. Construction of Ezh2-P2A-iCre transgenic founder mice

[0067] A. Extraction of the Ezh2-P2A-iCre plasmid from Example 2.

[0068] B. Preparation of fertilized egg donor female mice for superovulation and pseudopregnant female mice: Eight-week-old female mice were injected with pregnant mare serum gonadotropin (PMSG). Forty-eight hours after the PMSG injection, human chorionic gonadotropin (hCG) was injected. After the hCG injection, the female and male mice were housed together. At the same time, the female mice to be used for surrogacy were mated with vasectomized male mice to produce pseudopregnant female mice.

[0069] C. On the morning of the second day after the mice were put into the same cage, female mice were examined for plugs. Female mice with plugs were euthanized and fertilized eggs in the single-cell stage were collected. Male pronuclear microinjection was performed, injecting 30 μg of Ezh2-P2A-iCre plasmid, 15 μg of sgRNA (Example 1) and 30 μg of Cas9 protein.

[0070] D. The day after microinjection, the fertilized egg that has developed normally into a two-celled embryo was transferred into the uterus of a pseudopregnant female mouse. The mice born to the pseudopregnant female mouse are the F0 generation mice of the Ezh2-P2A-iCre transgenic gene.

[0071] Seven days after birth, the tails of the transgenic F0 generation mice were clipped, and their genomes were extracted. The transgenic mice were then identified using PCR. Mice that tested positive by PCR were identified as the founder mice of the Ezh2-P2A-iCre transgene.

[0072] 2. Establishment of Ezh2-P2A-iCre transgenic mice

[0073] A. The founder mice obtained above through the Ezh2-P2A-iCre transgenic process were crossed with wild-type mice, and the founder mice were passaged to establish a lineage, thus obtaining the F1 generation mice.

[0074] B. Extract DNA from the tails of F1 generation mice from different F0 generations, perform genotyping, select mice with stable genotypes as subsequent experimental strains, and the resulting mice are the transgenic tool mice.

[0075] Phenotypic analysis was performed on the Ezh2-P2A-iCre transgenic mice constructed in this example:

[0076] 1. Screening for Ezh2-P2A-iCre positive transgenic mice by PCR

[0077] (1) Primers

[0078] 5'arm PCR primer F1 (SEQ ID NO. 14): AGAGGGCTATCCAGACTGGTGAAG.

[0079] 5' PCR primer R1 (SEQ ID NO.15): TGCCAATGTGGATCAGCATTC.

[0080] 3' arm PCR primer F2 (SEQ ID NO.16): CACTGACTTTGACCAAGTCAGATC.

[0081] 3'armPCR primer R2 (SEQ ID NO. 17): GTAAGGGTTAGAGGTAAGGTGATACTG.

[0082] P2A-iCre PCR primer F3 (SEQ ID NO. 18): GGGAATGCTTCTAGCACAGGCT.

[0083] P2A-iCre PCR primer R3 (SEQ ID NO. 19): CACTGGTACAAACACTTTGCAGCT.

[0084] PCR product lengths: 5' arm 1916bp, 3' arm 1711bp, WT 1564bp.

[0085] (2) The reaction system is shown in Table 5.

[0086] Table 5 PCR reaction system

[0087] Serial Number name Amount added (μL) 1 2×TaqMasterMix, DyePlus, (VazymeP112-03) 12.5 2 Deionized water 9.5 3 PrimerF (10 pmol / μL) 1 4 PrimerR (10 pmol / μL) 1 5 Template (100ng / μL) 1

[0088] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles; extension for 5 min.

[0089] 2. Genotyping was performed using the PCR primers described above.

[0090] The results are as follows Figure 5 The 5' arm PCR product is 1916 bp, the 3' arm PCR product is 1711 bp, and the P2A-iCre PCR product is 1564 bp. Figure 5The bands below the numbers 4-8 correspond to the four F1 generation mice numbered 4-8, respectively. M represents the marker band, WT is the wild-type C57BL / 6JGpt, and N is the negative control. The results show that the F1 generation mice represented by numbers 4-8 are Ezh2-P2A-iCre transgenic positive mice.

[0091] 3. Cre-specific expression detection

[0092] Detection method: Ezh2-P2A-Cre mice were used in combination with CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to detect the expression of Ezh2 in the liver and the systemic expression of Ezh2-P2A-iCre mice. The results are shown in the table below. Figure 6 and Figure 7 Normally, CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato mice are green all over, which is unusual. However, this invention inserts LoxP sites on both sides of ZsGreen. When Cre recombinase is present, ZsGreen in the mouse genome is deleted, enabling the expression of tdTomato, which emits a dazzling red light.

[0093] Observation results: Observation of frozen tissue sections showed that ZsGreen and stop elements were excised from liver stem cells in mice that were offspring of CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato and Ezh2-P2A-iCre, and red fluorescent tdTomato was expressed. Figure 6 As shown, Ezh2 is co-localized with EpCAM and Bmi1. EpCAM is a marker of stem cells and naïve cells in various tissues, while Bmi1 is a stem molecule that maintains adult stem cells.

[0094] like Figure 7 As shown, Ezh2 is mainly expressed in adult stem cells.

[0095] This invention utilizes gene editing technology to create an Ezh2-P2A-iCre mouse model that specifically expresses Cre recombinase in adult stem cells under the drive of the Ezh2 promoter. This mouse model can be used to induce LoxP recombination in adult stem cells. For example, deleting the flux region, i.e., breeding this mouse model with a conditional knockout model mouse, can delete the gene segment between two LoxPs in adult stem cells.

[0096] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A gene expressed in liver stem cells Cre The method for constructing a tool mouse model is characterized by, Includes the following steps: (1) After mating between female and male mice, fertilized eggs in the single-cell stage were collected. Then, the male pronuclear microinjection vector, sgRNA and cas9 protein were injected. The fertilized eggs that developed normally into two cells were transplanted into the uterus of a pseudopregnant female mouse to obtain transgenic F0 generation mice. The F0 generation mice were identified and the founder mice of Ezh2-P2A-iCre transgene were screened. The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3; The transgenic vector includes the Ezh2-P2A-iCre fragment, the nucleotide sequence of which is shown in SEQ ID NO.7; (2) The founder mice of Ezh2-P2A-iCre transgenic were crossed with wild-type mice, and the F1 generation mice were obtained by passage and genotyping of the F1 generation mice to obtain the tool mouse model.

2. The construction method according to claim 1, characterized in that, In step (1), the method for preparing the sgRNA includes: a) The sgRNA coding region was obtained using the primer pair shown in SEQ ID NO.1-2, and the sgRNA coding region was ligated with the pUC57kan-T7 vector to obtain the sgRNA-pUC57kan-T7 plasmid; b) The sgRNA-pUC57kan-T7 plasmid was transcribed and purified in vitro to obtain the sgRNA.

Images