A method for constructing a TFRC humanized mouse model and its application

Abstract

The present invention relates to a method for constructing a TFRC humanized mouse model, comprising: (1) constructing a targeting vector expressing a humanized TFRC gene; (2) designing and obtaining a sgRNA targeting the mouse TFRC gene; (3) co-injecting or co-electroporating the targeting vector, sgRNA, and Cas9 protein into the cytoplasm or nucleus of a mouse fertilized egg, transplanting the fertilized egg into a pseudopregnant mouse, performing genotyping on the pseudopregnant offspring, and screening for positive F0 mice in which the correct humanized fragment was successfully inserted; and (4) breeding the F0 mice with background mice to obtain F1 mice, and screening for a TFRC humanized mouse model. The TFRC humanized mouse constructed by the present invention has application value in fields such as oncology and immunology.

Description

Technical Field

[0001] The present invention relates to the field of animal genetic engineering, and in particular to a method for constructing a TFRC humanized mouse model and its application. Background Art

[0002] As CRISPR / Cas9 technology matures, its applications are becoming more and more extensive. One of the most important applications is to use CRISPR / Cas9 to construct transgenic model animals (mainly gene-edited mice), thereby providing basic research on animal-level treatment before clinical practice.

[0003] Studying the pathogenesis of human diseases and identifying effective therapeutic drugs requires extensive preclinical testing. Due to ethical restrictions on the direct use of human cells and tissues for preclinical research, animal models have become an alternative for human biological research. Mice are small, easy to maintain and manipulate, have a short reproductive cycle, share genomic and physiological characteristics with humans, and have established gene modification technologies. Therefore, they have become a widely used mammalian model organism system. Currently, a considerable number of diseases are expected to be treated using CRISPR / Cas9 technology, validated through animal gene editing experiments, and subsequently applied to human clinical treatment.

[0004] The transferrin receptor (TFRC) (also known as CD71) is a transmembrane protein responsible for transporting iron absorbed from the digestive tract and released by erythrocyte degradation. During the terminal stages of erythrocyte differentiation, erythrocytes upregulate expression of the transferrin receptor (TFRC) gene to increase iron assimilation and heme production. TFRC interacts with transferrin (TF) on the cell surface to form a complex that is internalized through clathrin-mediated endocytosis. Once iron is released from mature endosomes, the iron-free TF-TFRC complex recycles to the cell surface for the next round of iron uptake. TFRC is highly expressed in actively proliferating cells, particularly tumor cells. Iron uptake, storage, transport, and regulation pathways are disrupted, leading to anemia in cancer patients, indicating that iron metabolism is closely linked to tumor cell survival. Studies have shown that anti-TFRC monoclonal antibodies can effectively inhibit the proliferation of hematologic malignancies. Furthermore, because iron metabolism plays a crucial role in the body's vital functions, TFRC deficiency can manifest as impaired erythrocyte development and abnormal iron metabolism. Therefore, the development of a TFRC Cas9-KI mouse model can be used to evaluate various studies related to the TFRC gene.

[0005] Mouse models play an irreplaceable role in drug development. However, due to racial differences, the efficacy of TFRC inhibitors screened in mice may differ from that in humans. Therefore, the development of a humanized TFRC mouse model is highly valuable for screening and evaluating drugs targeting the TFRC target. Currently, there are no reports on methods for constructing a humanized TFRC mouse model and its application in targeted drug development. Summary of the Invention

[0006] In response to the current problems, the first aspect of the present invention provides a method for constructing a TFRC humanized mouse model, which comprises the following steps:

[0007] (1) Constructing a targeting vector expressing the humanized TFRC gene for insertion of the humanized TFRC gene;

[0008] (2) Designing sgRNA targeting the translation start site of the mouse TFRC gene and obtaining the above sgRNA using in vitro transcription technology;

[0009] (3) co-injecting or co-electroporating the targeting vector constructed in step (1), the sgRNA obtained in step (2), and the Cas9 protein into the cytoplasm or nucleus of a mouse fertilized egg, transplanting the fertilized egg into a pseudo-pregnant mouse, and performing genotyping on the pseudo-pregnant offspring to screen for positive F0 mice in which the correct human fragment was successfully inserted;

[0010] (4) F0 mice were bred with background mice to obtain F1 mice. The F1 generation of mice were genetically identified and the TFRC humanized mouse model was selected.

[0011] Preferably, step (1) includes the following steps: according to the structure and function of human TFRC, inserting the human TFRC gene and TFRC 3'UTR-polyA after the mouse TFRC translation start codon (ATG), the selected human TFRC gene amino acid sequence is shown as SEQ ID No. 1, and the replaced mouse TFRC gene amino acid sequence is shown as SEQ ID No. 2.

[0012] Preferably, step (1) includes the following steps: selecting 91-760Aa of the human TFRC gene, replacing 91-763Aa of the mouse TFRC gene using homologous recombination technology, and the selected human TFRC gene sequence is shown in SEQ ID No.3.

[0013] Preferably, the targeting vector sequence successfully constructed in step (1) is shown as SEQ ID No. 4.

[0014] Preferably, the gene sequence of the sgRNA in step (2) is (a) SEQ ID NO.5 and SEQ ID NO.6, (b) SEQ ID NO.7 and SEQ ID NO.8, or (c) SEQ ID NO.9 and SEQ ID NO.10.

[0015] More preferably, the gene sequence of sgRNA in step (2) is SEQ ID NO.5 and SEQ ID NO.6.

[0016] Preferably, the strain of mice providing fertilized eggs and pseudo-pregnant mice in step (3) is BALB / c.

[0017] Preferably, the 5' end identification primers used for the F0 mouse genotype identification in step (3) are shown as SEQ ID NO.11 and SEQ ID NO.12, and the 3' end identification primers are shown as SEQ ID NO.13 and SEQ ID NO.14.

[0018] Preferably, the PCR reaction system used for the genotype identification of F0 mice in step (3) is as follows:

[0019]

[0020]

[0021] Preferably, the PCR reaction conditions used for the genotype identification of F0 mice in step (3) are as follows:

[0022]

[0023] The second aspect of the present invention provides the use of mice obtained by the above-mentioned construction method in studying the functions and mechanisms of action of the TFRC gene.

[0024] Preferably, the use is for non-diagnostic and non-therapeutic purposes.

[0025] A third aspect of the present invention provides the use of mice obtained by the above-mentioned construction method in screening drugs for treating diseases related to the TFRC gene.

[0026] Preferably, the use is for non-diagnostic and non-therapeutic purposes.

[0027] Beneficial effects of the present invention:

[0028] The animal model of the present invention is designed with a sgRNA that cleaves the murine TFRC gene. A donor containing the human TFRC gene, the TFRC gene's 3'UTR, and polyA is also designed. The sgRNA, donor, and Cas9 are mixed and injected into fertilized eggs of BALB / cJGpt mice. Homologous recombination occurs to generate positive F0 embryos. F0 mice are then bred with BALB / cJGpt mice to create a stable, genetically positive F1 mouse model. Compared to ES targeted mouse models, the animal model constructed in the present invention is highly efficient, rapid, simple, and inexpensive, saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the electrophoresis diagram of the 5' and 3' end gene identification results of TFRC-KI F0 mice;

[0030] Figure 2 This is the electrophoresis diagram of the 5' and 3' end gene identification results of TFRC-KI F1 mice;

[0031] Figure 3 Flow cytometric images of human TFRC expression in the bone marrow of BALB / c mice and BALB / c-hTFRC F1 heterozygous mice;

[0032] Figure 4 Flow cytometric plots of the ratio of peripheral blood B cells to T cells in BALB / c mice and BALB / c-hTFRC F1 heterozygous mice;

[0033] Figure 5 Flow cytometric plots of the proportions of NK cells and macrophages in peripheral blood of BALB / c mice and BALB / c-hTFRC F1 heterozygous mice;

[0034] Figure 6 Flow cytometric plots of the ratio of peripheral blood dendritic cells to neutrophils in BALB / c mice and BALB / c-hTFRC F1 heterozygous mice;

[0035] Figure 7 Flow cytometric plots showing the ratio of peripheral blood monocytes to eosinophils in BALB / c mice and BALB / c-hTFRC F1 heterozygous mice. DETAILED DESCRIPTION

[0036] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0037] Experimental Example 1: Establishment of a TFRC humanized mouse model

[0038] The present invention uses CRISPR / Cas9 technology to replace the murine TFRC gene with the human TFRC gene in BALB / c background mice, thereby constructing a mouse model that can express human TFRC. The specific method is as follows:

[0039] 1. Determine the human fragment replacement region and the inserted human sequence

[0040] Based on the structure and function of the TFRC gene, the human TFRC gene and its 3'UTR-polyA were inserted after the mouse TFRC translation start codon (ATG). The insert fragment is approximately 5.5 kb in length. The amino acid sequence of the selected human TFRC gene (Aa: 91-760) is shown in SEQ ID No. 1, and the amino acid sequence of the replaced mouse TFRC gene (Aa: 91-763) is shown in SEQ ID No. 2.

[0041] (SEQ ID No. 1)

[0042] (SEQ ID No. 2)

[0043] 2. Injection to obtain positive mice

[0044] 1) Determine the human fragment replacement region and the inserted human sequence

[0045] Based on the comparison of the extracellular functional domain of the human TFRC protein and the homology between humans and mice, 91-760Aa of the human TFRC gene replaced 91-763Aa of the mouse TFRC gene. The sequence of the selected human TFRC gene replacement is shown in SEQ ID No.3.

[0046]

[0047] 2) Construction of humanized targeting vector

[0048] The coding sequence of 91-760Aa of the human TFRC gene, the coding sequence of 1-90Aa of the mouse, and the 3'UTR of the human TFRC gene were constructed into a fusion CDS, and then constructed into a targeting vector. The homologous recombination technology was used to insert it into the starting position of the mouse TFRC gene. The sequence of the successfully constructed targeting vector is shown in SEQ ID No. 4 (the KI fragment is indicated in italics).

[0049]

[0050]

[0051]

[0052]

[0053] 3) Construction of sgRNA

[0054] (1) Synthesize sgRNA upstream and downstream primers, and purify the primers by PAGE;

[0055] (2) Dilute the upstream and downstream primers of sgRNA to 100 μmol / μl respectively, mix them in a 1:1 ratio, and slowly anneal at room temperature;

[0056] (3) The annealed double strands were ligated with Puc57-sgRNA-NEO-Amp (Bsa I) for 1 h, transformed, and coated on Amp+ plates;

[0057] (4) Pick single clones and perform PCR identification;

[0058] (5) PCR-positive single clones were further confirmed by sequencing using pUC57-T7-F as the sequencing primer;

[0059] (6) Using the correctly sequenced clone as a template, PCR amplifies the DNA product transcribed by sgRNA using primers;

[0060] (7) Using the DNA product transcribed from sgRNA as a template, sgRNA is transcribed and further purified.

[0061] 4) sgRNA screening in TFRC humanized mice

[0062] Three sets of sgRNAs (Tfrc-S1+Tfrc-S2, Tfrc-S3+Tfrc-S4, and Tfrc-S5+Tfrc-S6; see Table 1 for specific sequence information) were designed and synthesized. The sgRNA recognition site was located at the start codon of the mouse TFRC gene. Each pair of sgRNAs was then incubated with Cas9 protein and injected into 0.5-day-old fertilized eggs. After culturing to blastocysts, the knockout rate of the mouse TFRC gene was determined to verify the sgRNA cleavage activity.

[0063] sgRNA cutting experiment identification method: The collected blastocysts were PCR amplified. The PCR scheme is shown in Table 3-4. The amplified bands were sequenced by next-generation sequencing. The results were compared with the WT bands, and the probability of mutation was calculated (the identification scheme is shown in Table 2).

[0064] Table 1 sgRNA information and cutting efficiency

[0065]

[0066] 5) Establishment of TFRC humanized mouse model

[0067] The screened sgRNA (Tfrc-S1+Tfrc-S2) was used to design and construct an ssDNA donor carrying a human sequence. The ssDNA donor and Cas9 / sgRNA system were injected into 0.5-day-old mouse fertilized eggs and transplanted into 0.5-day-old pseudo-pregnant female mice. After the mice were born, the targeted mice (F0) were screened by genetic identification.

[0068] 6) Genotype identification of humanized F0 mice

[0069] The tail genomic DNA of the obtained F0 mice was subjected to post-target PCR identification using the two primer pairs shown in Table 2. The PCR reaction conditions and procedures are shown in Tables 3 and 4. Primers mTFRC-5tF1 / hTFRC-5tR1 are located outside the 5' homology arm and within the human fragment of the ssDNA donor, respectively. If this primer pair amplifies and produces a PCR product, it indicates that the target donor has been effectively inserted into the 5' end of the mouse genome; BGH-pA-tF1 / mTFRC-3tR1 are located within the human fragment of the ssDNA donor and outside the 3' homology arm, respectively. If this primer pair amplifies and produces a PCR product, it indicates that the target donor has been effectively inserted into the 3' end of the mouse genome.

[0070] Table 2 F0 identification primers

[0071]

[0072] Table 3 PCR reaction system

[0073]

[0074] Table 4 PCR reaction conditions

[0075]

[0076]

[0077] In this experiment, 49 F0 mice were injected and positive F0 mice were detected using the above identification scheme. Figure 1 As shown in the PCR electrophoresis results (WT is BALB / cJGpt genomic DNA (negative control); N is negative blank control (control without template); M is DNA Marker: TRANS2K PLUS II band: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), the 5' and 3' ends of the TFRC gene of mice #26 and #49 were both positive, and no mutations were found in the sequencing, indicating that the mice were positive mice with correct gene recombination and could be bred.

[0078] Positive F0 mice were bred with background mice to obtain F1, and the F1 generation mice were genetically identified. The results of the genetic identification of the F1 generation mice are shown in Figure 2 As shown (① is the 5' end, ② is the 3' end, and ③ is the wild type), mice #58, #60, and #64 all tested positive at both the 5' and 3' ends of the humanized TFRC gene, indicating that the resulting mice were heterozygous positive mice with correct genetic recombination. F1 mice were then extensively bred and interbred to obtain homozygous mice.

[0079] Experimental Example 2: Detection of hTFRC Expression in BALB / c-hTFRC F1 Heterozygous Mice

[0080] 1. Test method

[0081] Flow cytometry was used to examine TFRC protein expression in the bone marrow of BALB / c mice and BALB / c-hTFRC F1 heterozygous positive mice. Additionally, peripheral blood was collected from these mice and flow cytometry was used to analyze immune cell populations, including T cells, B cells, NK cells, macrophages, dendritic cells, neutrophils, monocytes, and eosinophils.

[0082] 2. Test results

[0083] like Figure 3As shown, human TFRC expression was detected in heterozygous BALB / c-hTFRC mice. In addition, the results of peripheral blood immune cell population analysis of wild-type and heterozygous BALB / c-hTFRC mice showed that the proportions of various immune cell subsets in humanized mice were consistent with those in wild-type mice ( Figure 4-7 ), indicating that the expression of humanized TFRC protein in mice has no effect on the development of immune cell subsets such as T cells, B cells, NK cells, macrophages, dendritic cells, neutrophils, monocytes, and eosinophils.

[0084] The above experimental results show that the present invention successfully constructed a BALB / c-hTFRC mouse model by replacing the mouse TFRC gene with a humanized gene, indicating that this model has broad application prospects in research in fields such as oncology and immunology.

[0085] Although the steps of this method have been described in detail, those skilled in the art will appreciate that modifications to some parameters and the overall scheme of the method are possible within the scope of this invention. Therefore, any changes, substitutions, or adjustments made within the spirit and principles of this invention are intended to be covered by this invention.

Claims

1. A method for constructing a TFRC humanized mouse model, characterized in that: The construction method comprises the following steps: (1) Constructing a targeting vector expressing the humanized TFRC gene for insertion of the humanized TFRC gene; (2) Designing sgRNA targeting the translation start site of the mouse TFRC gene and obtaining the sgRNA using in vitro transcription technology; (3) The targeting vector constructed in step (1), the sgRNA obtained in step (2), and the Cas9 protein are co-injected or co-electroporated into the cytoplasm or nucleus of a mouse fertilized egg, and the fertilized egg is transplanted into a pseudo-pregnant mouse. The pseudo-pregnant mice are genotyped and the positive F0 mice with the correct human fragment successfully inserted are screened; (4) F0 mice were bred with background mice to obtain F1 mice, and the F1 generation mice were genetically identified to screen for the TFRC humanized mouse model; The step (1) includes the following steps: according to the structure and function of human TFRC, inserting the human TFRC gene and TFRC 3'UTR-polyA after the mouse TFRC translation start codon (ATG), the selected human TFRC gene amino acid sequence is shown as SEQ ID No. 1, and the replaced mouse TFRC gene amino acid sequence is shown as SEQ ID No.

2.

2. The construction method according to claim 1, wherein The step (1) includes the following steps: selecting 91-760Aa of the human TFRC gene, and replacing 91-763Aa of the mouse TFRC gene using homologous recombination technology, wherein the selected human TFRC gene sequence is shown in SEQ ID No.

3.

3. The construction method according to claim 1, wherein The targeting vector sequence successfully constructed in step (1) is shown as SEQ ID No.

4.

4. The construction method according to claim 1, wherein The gene sequence of the sgRNA in step (2) is (a) SEQ ID NO.5 and SEQ ID NO.6, (b) SEQ ID NO.7 and SEQ ID NO.8, or (c) SEQ ID NO.9 and SEQ ID NO.

10.

5. The construction method according to claim 4, wherein: The gene sequences of sgRNA in step (2) are SEQ ID NO.5 and SEQ ID NO.

6.

6. The construction method according to claim 1, characterized in that The strain of mice providing fertilized eggs and pseudo-pregnant mice in step (3) is BALB / c.

7. The construction method according to claim 1, wherein: The 5' end identification primers used for the F0 mouse genotype identification in step (3) are shown as SEQ ID NO.11 and SEQ ID NO.12, and the 3' end identification primers are shown as SEQ ID NO.13 and SEQ ID NO.

14.

8. Use of mice obtained by the method for constructing a TFRC humanized mouse model according to any one of claims 1 to 7 in studying the functions and mechanisms of action of the TFRC gene.

9. Use of mice obtained by the method for constructing a TFRC humanized mouse model according to any one of claims 1 to 7 in screening drugs for treating diseases associated with the TFRC gene.

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