Construction method of Klf6 gene knockout mouse embryo model

Abstract

The invention discloses a construction method of a Klf6 gene knockout mouse embryo model, which comprises the following steps: S1, preparing a gene editing solution containing Cas9 enzyme and sgRNA for targeting a third exon region and a fourth exon region of a mouse Klf6 gene by utilizing a CRISPR / Cas9 gene editing technology; s2, taking out the fertilized eggs of the mouse, transfecting the fertilized eggs of the mouse with a gene editing solution, and then performing in-vitro culture on the fertilized eggs; s3, carrying out embryo transplantation on the two-cell stage embryo obtained in the step S2 into a pregnant female mouse, and then carrying out conventional feeding; and S4, 7-8 days after embryo transplantation, taking out the uterus and stripping the embryo to obtain the Klf6 gene knockout mouse embryo model. According to the method, gene knockout is carried out by using CRISPR / Cas9 in the fertilized egg period in combination with an electrotransfection method, and compared with an existing method, the construction process time can be greatly shortened, the working efficiency is obviously improved, and the success rate is high.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for constructing a mouse embryo model with Klf6 gene knockout. Background Technology

[0002] The successful conception of a new life begins with the healthy development of the embryo. Early pregnancy, especially between weeks 5 and 12, is a high-risk window for miscarriage. After 12 weeks, the risk drops significantly to approximately 1%. However, clinical data on miscarriages before 5 weeks of gestation are lacking due to the small size of the embryo, although the estimated miscarriage rate is as high as 50%. Various factors are known to cause miscarriage, including gamete and embryonic chromosomal abnormalities, uterine structural problems, and autoimmune diseases. Of concern, the cause remains unknown in more than half of recurrent miscarriage cases, with embryonic developmental abnormalities currently considered a potential contributing factor.

[0003] Because mouse embryos and human embryos share similar developmental stages and gene regulatory networks, and because human embryo research faces ethical limitations, mouse embryo models are typically chosen as an alternative system for human embryo research. The gastrulation stage in mouse embryos corresponds to the third week of human embryonic development and is a critical period. During this time, the embryo remodels the homogeneous cell population of the blastocyst stage into the ectoderm (Ect), mesoderm (Mes), and endoderm (End) with a defined spatial arrangement through complex cell migration and fate determination mechanisms, laying the foundation for the formation of subsequent organelle primordia. The formation and differentiation of the three germ layers are finely regulated by a complex network, including the spatiotemporal specific activation of key signaling pathways such as Wnt, Nodal, and BMP, and also relying on the synergistic effects of lineage-specific transcription factors. The absence of specific transcription factors leads to severe germ layer developmental disorders, resulting in embryonic developmental failure. For example, the absence of the mesoderm-specific gene Brachyury results in the absence of the tail mesoderm and embryonic lethality. Therefore, exploring the regulation of embryonic trilaminar differentiation by transcription factors is of great significance for understanding early pregnancy miscarriage.

[0004] The Krüppel-like factor family (KLF family) is an important transcriptional regulator. Its members specifically recognize and bind to target gene regulatory regions through three highly conserved tandem C2H2 zinc finger domains at the C-terminus. Studies have shown that the KLF family plays an irreplaceable regulatory role in embryonic development; the absence of any member can lead to severe developmental defects or even embryonic lethality. For example, Klf1 gene knockout results in impaired erythropoiesis in mouse embryos, leading to embryonic lethality on day 14 (E14); the absence of transcription factor SP1 causes embryonic developmental delay, resulting in embryonic lethality at E11. Homozygous mutants of transcription factor KLF6 exhibit significant developmental defects at E10.5, with failure of the embryonic hematopoietic and extracellular hematopoietic systems, incomplete abdominal turning and body axis development, subsequently leading to embryonic lethality at E12.5. Existing research has clarified the important role of KLF6 in late embryonic development; however, whether it ensures normal embryonic development by regulating early differentiation of the three germ layers remains unclear.

[0005] Existing methods for constructing Klf6 gene knockout mouse embryos involve viral infection of embryonic stem cells, creating chimeras at the blastocyst stage, and then obtaining knockout phenotype embryos through chimeric hybridization. However, this method typically takes several months and is inefficient. A faster and more efficient method for constructing Klf6 gene knockout embryo models is needed to meet research requirements. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing a method for constructing a mouse embryo model with Klf6 gene knockout.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] The first aspect of the present invention provides a method for constructing a Klf6 gene knockout mouse embryo model, comprising the following steps:

[0009] S1. Using CRISPR / Cas9 gene editing technology, prepare a gene editing solution containing Cas9 enzyme and sgRNA for targeting the third and fourth exon regions of the mouse Klf6 gene;

[0010] S2. Remove mouse fertilized eggs, transfect the mouse fertilized eggs with the gene editing solution, and then culture the fertilized eggs in vitro.

[0011] S3. The two-cell stage embryos obtained in step S2 are transferred into surrogate female mice, and then they are fed in a routine manner.

[0012] S4. Seven to eight days after embryo transfer, the embryo is removed from the uterus and separated to obtain a mouse embryo model with the Klf6 gene knocked out.

[0013] The sgRNA includes sgRNA1, sgRNA2, sgRNA3, and sgRNA4, whose sequences are shown in SEQ ID NO.1~4 in sequence.

[0014] In step S1, sgRNA1, sgRNA2, sgRNA3, and sgRNA4 are mixed in equal molar amounts in the gene editing solution.

[0015] Further, in step S2, the fertilized eggs were transfected by electroporation using gene editing solution: fertilized eggs from female mice were taken out, digested with hyaluronidase at 35-38℃, washed in M2 culture medium, digested with acidic styrosine solution, and then transferred to the prepared gene editing solution. After mixing, the eggs were transferred to an electroporation cuvette and electroporated 5 times at 30V and 3ms. After electroporation, the fertilized eggs were washed in M2 culture medium and then transferred to a drop of KSOM culture medium that had been equilibrated in a 37℃ 5% CO2 incubator and cultured overnight.

[0016] Further, in step S3, well-developed two-cell stage embryos are selected from the embryos cultured in vitro in step S2, placed in M2 culture medium at 35~38℃ for equilibration, and then transplanted into surrogate female mice.

[0017] Furthermore, the construction method of the present invention also includes step S5, embryo genotype identification: take the embryo obtained in step S4, extract DNA, and use PCR amplification detection.

[0018] Furthermore, the primers for PCR amplification are outer primers and inner primers;

[0019] The upstream sequence of the outer primer is: SEQ ID NO.5: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence is SEQ ID NO.6: 5'-CTGCGAGTGTCTCAAGCGA-3';

[0020] The upstream sequence of the inner primer is SEQ ID NO.7: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence is SEQ ID NO.8: 5'-CCCAGACCATGCACGACTT-3'.

[0021] Furthermore, if the outer primer amplifies a band of 780-1080 bp and the inner primer shows no amplification band, it indicates that the embryo is Klf6. - / - Embryo.

[0022] A second aspect of the present invention provides the application of the Klf6 gene knockout mouse embryo model obtained by the above-described construction method in the study of embryonic trilaminar differentiation.

[0023] The beneficial effects of this invention are as follows: Existing research has clearly established the important role of Klf6 in the later stages of embryonic development, but there is a lack of efficient methods for constructing Klf6 knockout embryo models for embryonic differentiation research. This invention uses CRISPR / Cas9 combined with electroporation to knock out the gene during the fertilized egg stage. Compared with current methods for constructing Klf6 knockout embryo models, this method can greatly shorten the construction time, significantly improve work efficiency, and has a high success rate. It can be used to study the association between Klf6 and the differentiation of the three germ layers of the embryo, and can be applied to clinical research on early pregnancy miscarriage. Attached Figure Description

[0024] Figure 1 The expression of Klf6 in the three germ layers was detected by immunofluorescence.

[0025] Figure 2 This is a schematic diagram of the sgRNA target site of Klf6 and the primer sites for PCR detection and amplification.

[0026] Figure 3 This is a lipoglycogel electrophoresis image for embryo genotyping.

[0027] Figure 4 This is the result of RNA-seq genotyping.

[0028] Figure 5 These are the results of differentially expressed gene analysis.

[0029] Figure 6 These are the results of GO enrichment analysis of differentially expressed genes in the epidermis and mesoderm. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0032] Example 1

[0033] I. Immunofluorescence confirmation of Klf6 expression in the three germ layers

[0034] Male C57BL / 6J mice (8-10 weeks old) and female C57BL / 6J mice (6-8 weeks old) were housed together (1:1). Vaginal plugs were examined between 8:00-9:00 AM the following morning. Female mice with plugs were euthanized on day 7.5 using cervical dislocation, and embryos were isolated. Embryos were fixed in 4% paraformaldehyde at room temperature for 30 minutes. After washing three times with 200 μL of PBS, 200 μL of 1% BSA diluted KLF6 rabbit polyclonal antibody (Wuhan Sanying 14716-1-AP, 1:200 dilution) was added, and incubated overnight at 4°C. The next day, after washing three times with PBS, 200 μL of PBS diluted fluorescently labeled secondary antibody (FITC-labeled goat anti-rabbit IgG, 1:200 dilution) was added, and incubated in the dark for 4 hours. Finally, after washing with PBS and staining with DAPI (1:200 PBS dilution) for 5 minutes, the results were photographed and recorded using a confocal microscope (Nikon). Figure 1 Immunofluorescence showed that KLF6 was distributed in all components of mouse embryos on day 7.5 and had obvious co-localization with DAPI, indicating that KLF6 is mainly located in the cell nucleus.

[0035] II. Design of sgRNA

[0036] Based on the mouse Klf6 gene sequence (NCBI ID: NC_000079.7), four sgRNA sequences were designed using the online CRISPR target design tool on the IDT website. The target locations are as follows: Figure 2 As shown, sgRNA1 and sgRNA2 target the first 150bp and 56bp positions of the third exon of the Klf6 gene, respectively, while sgRNA3 and sgRNA4 target the last 500bp and 700bp positions of the fourth exon of the Klf6 gene, respectively. The four sgRNA sequences are as follows:

[0037] sgRNA1 (SEQ ID NO.1): 5'-TGGTACCTTTGGCAAGCTAA-3';

[0038] sgRNA2 (SEQ ID NO.2): 5'-TCACTGACAAGATAGGGTAA-3';

[0039] sgRNA3 (SEQ ID NO.3): 5'-ACACCTGGATGAGTCCCATC-3';

[0040] sgRNA4 (SEQ ID NO. 4): 5'-CAAGATTGCTTCTCCTCACG-3'".

[0041] III. Design of primers for verification

[0042] Two pairs of primers were designed using the NCBI Primer designing tool website for PCR amplification.

[0043] The upstream sequence of the outer primer was SEQ ID NO.5: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence was SEQ ID NO.6: 5'-CTGCGAGTGTCTCAAGCGA-3'; the upstream sequence of the inner primer was SEQ ID NO.7: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence was SEQ ID NO.8: 5'-CCCAGACCATGCACGACTT-3'. The amplified products were detected by 1% agarose gel electrophoresis.

[0044] The outer primer amplifies the third and fourth exon regions of the complete Klf6 gene. - / - Because the third and fourth exon regions were completely knocked out, the length of the amplified fragment by the outer primers was shortened, while the downstream sequence of the inner primers was located within the designed knockout region, Klf6. - / - Since the third and fourth exon regions are completely knocked out, the inner primers will not be able to amplify the signal.

[0045] IV. Preparation of Gene Editing Solution

[0046] Take 1 μL of each of the four 2 μM / μL sgRNA solutions mentioned above, add 1 μL of Cas9 protein and 5 μL of 2× electroporation buffer, mix well, and place in a PCR instrument. React according to the program of 37℃ for 15 min. After the reaction, add an equal volume of Opti-MEM culture medium to prepare the gene editing solution.

[0047] V. Electroporation transfection of fertilized eggs

[0048] Four-week-old female C57BL / 6J mice were selected and injected intraperitoneally with 10U of pregnant mare serum gonadotropin (PMSG) between 15:00 and 17:00. 46-47 hours later, 10U of human chorionic gonadotropin (hCG) was injected, and the mice were then caged with eight-week-old male C57BL / 6J mice at a 1:1 ratio.

[0049] Prepare embryo culture droplets: 5-6 hours before gene editing, prepare several 20ul KSOM culture droplets in a 35mm culture dish, cover with mineral oil, and place in a 37℃ 5% CO2 incubator for equilibration for 5-6 hours.

[0050] Electroporation transfection of fertilized eggs: 12 hours after mating, check the vaginal plugs of superovulated female mice. The presence of a vaginal plug indicates successful mating. After anesthesia, euthanize the female mouse with the plug by cervical dislocation. Open the abdominal cavity, remove the enlarged oviduct from the ampulla, and place it in a drop of equilibrated M2 culture medium at 37°C. Under a stereomicroscope, use watch forceps to tear open the enlarged part of the oviduct to release the fertilized egg mass. Then, place the egg mass in hyaluronidase at 37°C for 2 minutes for digestion. Wash the fertilized eggs three times in M2 culture medium, digest the zona pellucida with acidic styrofoam (reducing it by 1 / 3), and transfer the fertilized eggs into prepared gene editing medium. After mixing, transfer to an electroporation cuvette and electroporate five times at 30V and 3ms. After electroporation, wash the fertilized eggs three times in M2 culture medium and transfer them into a drop of KSOM culture medium pre-equilibrated in a 37°C incubator for overnight incubation at 37°C with 5% CO2.

[0051] VI. Two-cell embryo transfer

[0052] On the afternoon of the day the fertilized eggs were retrieved, naturally estrus surrogate female mice were caged with vasectomized male mice at a ratio of 1:2. Surrogate female mice with ovarian endocardium (plugs) were ready for embryo transfer 12 hours later. Well-developed two-cell stage embryos were selected from the embryos cultured in vitro the previous day and placed in M2 culture medium at 37°C for equilibration. After anesthetizing the surrogate female mice with ovarian endocardium via intraperitoneal injection, alcohol was sprayed onto their backs. An incision was made in the middle of their backs, and the fat next to the ovary was gently grasped with forceps. The fallopian tube was pulled out through the incision. A small hole was made 2 cm upstream of the dilated part of the fallopian tube using an insulin syringe. The embryo was gently blown into the fallopian tube using a glass transfer needle. The fallopian tube was then returned to the body cavity, and the wound was clamped shut. The female mice after embryo transfer were housed in an independent cage ventilation system (IVC).

[0053] VII. Separation of germ layers in mouse embryos on day 7.5

[0054] Seven days after embryo transfer, at noon, mouse embryos on day 7.5 were retrieved and the germ layers were separated. The surrogate female mouse was euthanized under anesthesia, the abdominal cavity was opened, the uterus was removed and placed in DMEM medium. Under a stereomicroscope, the embryo was dissected with clockwise forceps, and the embryo was cut along the boundary between the embryonic region and the extraembryonic region using a glass needle. The cut embryo was placed in droplets of trypsin and trypsin solution and incubated on ice for 10 minutes. The embryo was then transferred to DMEM medium containing FBS, and the ecto, mesoderm, and endoderm were separated and collected using a pipette slightly larger than the embryo's diameter.

[0055] VIII. Embryo Genotyping

[0056] DNA was extracted from a small amount of embryonic tissue using DNA lysis buffer. Using the embryonic DNA as a template, PCR amplification was performed using the aforementioned verification primers. The reaction mixture consisted of 1 μL DNA template, 5 μL PCR enzyme, 3 μL enzyme-free water, 0.5 μL upstream primer, and 0.5 μL downstream primer. The reaction conditions were 98℃ for 30 seconds; (98℃ for 15 seconds, 62℃ for 15 seconds, 72℃ for 45 seconds) × 32 cycles; 72℃ for 5 minutes. The amplified products were identified by 1% agarose gel electrophoresis (e.g., ...). Figure 3 The marker bands are 200bp, 500bp, 800bp, 1000bp, 1500bp, 2000bp, and 4000bp, wild type (Klf6). + / + The outer primer amplifies a fragment of 5300 bp, and the inner primer band is 2200 bp; the knockout type (Klf6) - / - The outer primers amplified fragments of 780-1080 bp in length, while the inner primers showed no bands; heterozygotes (Klf6) + / - The outer primer amplification simultaneously detected fragments of 5300bp and 780-1080bp, while the inner primer band was 2200bp.

[0057] IX. RNA-seq for Genotyping and Differentially Expressed Gene Analysis

[0058] Add 0.2% Triton X-100 solution to the collected germ layer samples to extract mRNA. After reverse transcribing the collected mRNA into cDNA, construct a library according to the Vazyme kit TD502 instructions and send the constructed library to a sequencing company for sequencing.

[0059] The FASTQ files returned from sequencing were spliced ​​and aligned using TopHat v2.0.11, with the mm9 genome as the reference genome. An index was built using Samtools Index software, and the indexed BAM files were converted to BigWig format using BamCoverage, followed by normalization based on FPKM (Fragments Per Kilobase Per Million Mapped Reads). The generated BigWig files were uploaded to the UCSC Genome Browser for visualization, where Klf6 was visible. - / - The signal peaks of the third and fourth exons of the embryo disappear, Klf6 + / + Embryonic signal peaks are still present. Figure 4 This indicates that the Klf6 knockout mouse embryo model was successfully constructed.

[0060] Based on FPKM values, differentially expressed genes were screened according to FPKM greater than 1 and absolute difference greater than 2. After Klf6 knockout, 1191 genes were downregulated and 846 genes were upregulated in the ectoderm; 1372 genes were downregulated and 860 genes were upregulated in the mesoderm; and 187 genes were downregulated and 172 genes were upregulated in the endoderm. Figure 5 This indicates that Klf6 plays a more significant role in gene expression regulation in the ectoderm and mesoderm. Using DAVID v2025 (https: / / david.ncifcrf.gov / ), gene ontology (GO) enrichment analysis of differentially expressed genes in the ectoderm and mesoderm revealed that, after Klf6 deletion, downregulated genes in the ectoderm were mainly associated with biological processes such as amino acid metabolism, purine compound metabolism, signal transduction, and tRNA modification, while upregulated genes in the ectoderm were mainly associated with biological processes such as DNA damage repair, inhibition of nervous system development, and inhibition of transcription. In the mesoderm, downregulated genes were mainly associated with biological processes such as galactosylation, enzyme activity regulation, signal transduction, and vesicle-mediated transport, while upregulated genes in the mesoderm were mainly associated with biological processes such as inhibition of cardiovascular system development and inhibition of epithelial cell differentiation. Figure 6 This indicates that the transcription factor KLF6 can coordinate metabolism and material exchange in the embryo on day 7.5, participate in the formation of a core regulatory network in the ectoderm and mesoderm to maintain developmental homeostasis, and ensure normal embryonic development.

Claims

1. A method for constructing a Klf6 gene knockout mouse embryo model, characterized in that, Includes the following steps: S1. Using CRISPR / Cas9 gene editing technology, prepare a gene editing solution containing Cas9 enzyme and sgRNA for targeting the third and fourth exon regions of the mouse Klf6 gene; S2. Remove mouse fertilized eggs, transfect the mouse fertilized eggs with the gene editing solution, and then culture the fertilized eggs in vitro. S3. The two-cell stage embryos obtained in step S2 are transferred into surrogate female mice, and then they are fed in a routine manner. S4. 7.5 to 8.0 days after embryo transfer, the embryo is removed from the uterus and detached, thus obtaining the Klf6 gene knockout mouse embryo model.

2. The construction method according to claim 1, characterized in that: The sgRNA includes sgRNA1, sgRNA2, sgRNA3, and sgRNA4, whose sequences are shown in SEQ ID NO. 1~4 respectively.

3. The construction method according to claim 2, characterized in that: In step S1, sgRNA1, sgRNA2, sgRNA3, and sgRNA4 are mixed in equal molar amounts in the gene editing solution.

4. The construction method according to claim 1, characterized in that: In step S2, fertilized eggs were transfected by electroporation using gene editing solution: fertilized eggs from female mice were removed, digested with hyaluronidase at 35-38℃, washed in M2 culture medium, and digested with acidic styrofoam solution. The fertilized eggs were then transferred to the prepared gene editing solution, mixed, and transferred to an electroporation cuvette. They were electroporated 5 times at 30V and 3ms. After electroporation, the fertilized eggs were washed in M2 culture medium and transferred to a drop of KSOM culture medium that had been pre-equilibrated in a 37℃ 5% CO2 incubator. The mixture was then incubated overnight.

5. The construction method according to claim 1, characterized in that: In step S3, well-developed two-cell stage embryos are selected from the embryos cultured in vitro in step S2, placed in M2 culture medium at 35~38℃ for equilibration, and then transplanted into surrogate female mice.

6. The construction method according to claim 1, characterized in that: It also includes step S5, embryo genotype identification: take the embryo obtained in step S4, extract DNA, and use PCR amplification for detection.

7. The construction method according to claim 6, characterized in that: The primers for PCR amplification are outer primers and inner primers; The upstream sequence of the outer primer is: SEQ ID NO.5: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence is SEQ ID NO.6: 5'-CTGCGAGTGTCTCAAGCGA-3'; The upstream sequence of the inner primer is SEQ ID NO.7: 5'-ACTGGCAGCACAACATCTGG-3', and the downstream sequence is SEQ ID NO.8: 5'-CCCAGACCATGCACGACTT-3'.

8. The construction method according to claim 7, characterized in that: If the outer primer amplifies a band of 780-1080 bp and the inner primer does not amplify a band, it indicates that the embryo is Klf6. - / - Embryo.

9. The application of the Klf6 gene knockout mouse embryo model obtained by the construction method according to any one of claims 1 to 8 in the study of embryonic trilaminar differentiation.

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