Humanized FKBP8 gene knock-in animal model and construction method therefor
By using CRISPR/Cas9 technology to perform homologous recombination in Exon2 and Exon8 of the mouse FKBP8 gene, a humanized FKBP8 gene knock-in animal model was constructed, which solved the problem that it is difficult to study the FKBP8 gene in clinical diseases in existing technologies, and realized the research and screening effects in animal models.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2026-04-02
AI Technical Summary
Current technology has not yet successfully constructed a humanized FKBP8 gene knock-in animal model, making it difficult to study the role of the FKBP8 gene in the pathogenesis and treatment of clinical diseases.
Using CRISPR/Cas9 technology, sgRNAs were designed to undergo homologous recombination in Exon2 and Exon8 of the mouse FKBP8 gene to construct a humanized FKBP8 gene knock-in animal model. The humanized FKBP8 gene knock-in animals were obtained by microinjecting Cas9/sgRNA into the fertilized eggs of donor animals and by PCR and Southern blot identification.
A humanized FKBP8 animal model was successfully established, enabling the screening of drugs targeting the FKBP8 gene and its products, as well as the study of their related pathogenesis and treatment mechanisms, in the animal model.
Smart Images
Figure CN2024121330_02042026_PF_FP_ABST
Abstract
Description
A humanized FKBP8 gene knock-in animal model and a construction method thereof TECHNICAL FIELD
[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a humanized FKBP8 gene knock-in animal model and a construction method thereof. BACKGROUND
[0002] FKBP8 belongs to the FK506 binding protein (FKBPs) family, and members of the protein family act as peptidyl prolyl cis-trans isomerases (PPIases) to play a role in immune regulation and basic cellular processes involving protein folding and transport, and are involved in biological processes such as neuron fate, mitochondrial autophagy, and apoptosis regulation.
[0003] Mice have a short life cycle and genetic similarity to humans, and are the most in-depth mammalian model organisms for genomics, transcriptomics, proteomics, and phenotype research, and are usually the preferred choice as mammalian model organisms. Sequence alignment using clustalw shows that the FKBP8 genes of mice and humans are highly non-conservative (with a conservation of only 37.96%), and more and more studies have found that the FKBP8 gene and its product are important in clinical diseases, so it is necessary to construct a human FKBP8 gene knock-in animal model to allow researchers to further study the role of the FKBP8 gene and its product in the pathogenesis and treatment of related clinical diseases.
[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism used by bacteria to degrade invading viral DNA and other foreign DNA. The CRISPR / Cas9 system derived from Streptococcus pyogenes is currently the most commonly used one. The CRISPR / Cas9 technology has been successfully used in bacteria, yeast, plants, fish, and mammals, and is the most effective gene editing technology to date. There is currently no related research on a humanized FKBP8 gene knock-in animal model.
[0005] SUMMARY
[0006] The purpose of the present application is to construct a humanized FKBP8 gene knock-in animal model for studying the pathogenesis and therapeutic targets of human disease models.
[0007] To this end, the present application provides a construction method of a humanized FKBP8 gene knock-in animal model, comprising the following steps:
[0008] (1) constructing a targeting vector for humanized FKBP8 gene knock-in, and transfecting the targeting vector, Cas9, and sgRNA into a donor animal zygote;
[0009] (2) transplant the surviving fertilized egg cell into the oviduct of the pseudo-pregnant animal to obtain F0 generation animals, identify the F0 generation animals, and the animals showing positive homologous recombination are chimeric animal;
[0010] (3) cross the chimeric animal with a wild-type animal to obtain F1 generation animals and identify them, and the F1 animals showing positive homologous recombination are humanized FKBP8 gene knock-in animals.
[0011] Specifically, the above step (1) is specifically: designing a targeting strategy based on the EGE system developed by CRISPR / Cas9, using the extracellular region of the human FKBP8 gene to replace the extracellular region of the FKBP8 gene of the donor animal, and designing sgRNAs in the Exon2 and Exon8 of the FKBP8 gene of the donor animal; design sgRNAs at the 5' end and 3' end target region, detect in vitro activity, select sgRNAs in combination with sgRNA specificity and cutting position, transcribe sgRNAs and Cas9 in vitro, prepare RNA for microinjection; according to the targeting scheme, construct a recombinant targeting vector, and transfect the recombinant targeting vector, Cas9, and sgRNAs into the fertilized egg of the donor animal.
[0012] Specifically, in the above step (1), four sgRNAs are designed at the 5' end target region, and the sequences are shown in SEQ ID NO. 1-4; four sgRNAs are designed at the 3' end target region, and the sequences are shown in SEQ ID NO. 5-8; the 5' target site and the 3' target site with the highest activity are selected for the next experiment.
[0013] Specifically, in the above step (1), the 5' target site EGE-HJL-0170-A-sgRNA3 with the highest activity has the sequence shown in SEQ ID NO. 3; and the 3' target site EGE-HJL-0170-A-sgRNA7 with the highest activity has the sequence shown in SEQ ID NO. 7.
[0014] Specifically, in the above step (1), the sgRNAs are detected for in vitro activity by UCA TM
[0015] Specifically, in the above step (2), PCR is used to identify the genotype of the F0 generation animals; the primer sequences are shown in SEQ ID NO. 9-12.
[0016] Specifically, in the above step (3), PCR and Southern blot are used to identify the F1 generation animals; the DNA of the F1 generation animals identified as positive by PCR is detected by Southern blot and sequencing to confirm correct recombination and no random insertion, and the humanized FKBP8 gene knock-in animal model is successfully constructed.
[0017] Specifically, the animal is a mouse.
[0018] Compared with the prior art, the present application has the following advantages and beneficial effects:
[0019] The construction method of the humanized FKBP8 gene knock-in animal model provided by the present application replaces the FKBP8 gene sequence of the donor animal with the human FKBP8 gene sequence to achieve the purpose of humanizing the FKBP8 gene of the donor animal, successfully establishes a humanized FKBP8 animal model, and can screen drugs targeting the FKBP8 gene and its products or research the pathogenesis and treatment mechanism related to the FKBP8 gene and its products in the animal model. BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 is a targeting strategy for preparing a FKBP8 humanized knock-in mouse model based on the EGE system developed by CRISPR / Cas9.
[0021] FIG. 2 is a Southern blot screening strategy for F1 generation positive mice.
[0022] FIG. 3 is the activity detection result of Cas9 / sgRNA.
[0023] FIG. 4 is an enzyme digestion identification and sequencing scheme.
[0024] FIG. 5 is a targeting vector map.
[0025] FIG. 6 is a primer design strategy for F0 generation mice.
[0026] FIG. 7 is the tail genotype identification result of F0 generation mice (Primers: EGE-HJL-0170-A-L-GT-F / EGE-HJL-0170-A-L-GT-R).
[0027] FIG. 8 is the tail genotype identification result of F0 generation mice (Primers: EGE-HJL-0170-A-R-GT-F / EGE-HJL-0170-A-R-GT-R).
[0028] FIG. 9 is the tail genotype identification result of F1 generation mice (Primers: EGE-HJL-0170-A-L-GT-F / EGE-HJL-0170-A-L-GT-R).
[0029] FIG. 10 is the tail genotype identification result of F1 generation mice (Primers: EGE-HJL-0170-A-R-GT-F / EGE-HJL-0170-A-R-GT-R).
[0030] FIG. 11 is the Southern blot detection result of F1 generation mice. DETAILED DESCRIPTION
[0031] The technical solutions in the present application will be described clearly and completely in connection with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Although the representative embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present application without departing from the scope of the present application. Therefore, the scope of the present application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.
[0032] The effects of the humanized FKBP8 gene knock-in animal model and the construction method thereof of the present application are studied through specific embodiments.
[0033] Embodiment 1:
[0034] 1. Design of targeting vector
[0035] The human FKBP8 gene is on the reverse strand of chromosome 19 (NC_000019.9_c18654383-18642573, 11811 bp), and the expressed protein is 412 aa in length, with 14 transcripts. In this embodiment, transcript 201 is referred to. The mouse FKBP8 gene is on the positive strand of chromosome 8 (NC_000074.7_70980371-70987978, 7608 bp), and the expressed protein is 402 aa in length, with 11 transcripts. In this embodiment, transcript 202 is referred to.
[0036] After homology comparison, the sequence difference between human and mouse FKBP8 genes is accurately found. There are differences in Exon2 and Exon8 regions. After repeated groping and adjustment, it is found that the extracellular region of human FKBP8 gene can be used to replace the extracellular region of mouse FKBP8 gene, which has good homologous recombination efficiency, realizes the purpose of high-efficiency expression of human sequence under the mouse promoter, and more importantly, can realize the function of human FKBP8 gene on the mouse model.
[0037] The EGE system developed by Bio-Smart based on CRISPR / Cas9 was used to prepare gene humanization knock-in mode mice. sgRNAs were designed in Exon2 and Exon8 of FKBP8 gene of C57BL / 6J mice, respectively. The actual sizes of the 5' end homologous arm and the 3' end homologous arm of the targeting vector for homologous recombination were 1.2 kb and 1.2 kb, respectively. The targeting strategy is shown in Figure 1.
[0038] 2. Cas9 / sgRNA design
[0039] Based on the design principle of sgRNA, four sgRNAs were designed in the 5' target site and 3' target site region, respectively, and the corresponding target sequences are shown in Table 1.
[0040] Table 1 sgRNA sequence (SEQ ID NO. 1-8)
[0041] The DNA sequences of the target genes of different strains may be different. In order to ensure the correctness of the designed Cas9 / sgRNA, the target gene DNA sequence of C57BL / 6J mouse tail was first amplified by PCR and sequenced, and the verification results showed that the sgRNA target sequence was completely consistent with the standard sequence given by Genebank database.
[0042] UCA TM The in vitro activity detection of sgRNA has the advantages of no species restriction, high throughput, wide adaptability, high sensitivity and simplicity. The sgRNA was detected by UCA TM activity, combined with the specificity and cleavage position of sgRNA, EGE-HJL-0170-A-sgRNA3 (SEQ ID NO. 3) and EGE-HJL-0170-A-sgRNA7 (SEQ ID NO. 7) were selected for the next experiment, and the detection results are shown in Figure 3.
[0043] 3. RNA preparation of Cas9 / sgRNA
[0044] EGE-HJL-0170-A-sgRNA3 and EGE-HJL-0170-A-sgRNA7 and Cas9 were transcribed in vitro to prepare RNA for microinjection.
[0045] 4. Construction of targeting vector
[0046] According to the targeting scheme, the targeting vector was constructed, and the enzyme digestion identification and sequencing (Figure 4) were carried out to confirm that the targeting vector was constructed correctly, and the prepared targeting vector for microinjection is shown in Figure 5.
[0047] 5. Microinjection
[0048] Cas9 / sgRNA and targeting vector were microinjected into mouse zygotes, and the birth of F0 mice after injection was as follows:
[0049] 6. Genotype identification of F0 mice
[0050] Since the early embryo cleavage speed is very fast, the F0 mice obtained are chimeras. Therefore, the F0 genotype obtained by identifying the F0 mouse tail is only for reference, and cannot represent that it is a heritable gene mutant type. The heritable genotype needs to be determined after the F1 mouse tail is detected. The F0 mouse identification primer and results are shown in Tables 2-3 and Figures 6-8.
[0051] Table 2 primer information (SEQ ID NO. 9-12)
[0052] and Table 3 genotype identification PCR conditions
[0053] Through PCR amplification and product sequencing, it is shown that E4L0170-0001, E4L0170-0015, E4L0170-0017, E4L0170-0018 and E4L0170-0022 are positive F0 mice.
[0054] 7, mating to obtain F1 generation mice
[0055] The F0 mice identified as positive are mated with wild type mice to obtain F1 mice with stable genotype, and the mating results are as follows (showing the F1 mice obtained by mating the positive F0 mice with wild type mice respectively):
[0056] In order to screen the gene targeting mice with correct recombination, PCR and Southern blot methods are used to verify the F1 mice.
[0057] The PCR primer design principle is the same as that for identifying F0 mice, and the identification results are shown in Figures 9-10. PCR amplification shows that 1E4L0170-0001, 1E4L0170-0008, 1E4L0170-0009, 1E4L0170-0012, 1E4L0170-0013, 1E4L0170-0016, 1E4L0170-0018, 1E4L0170-0020, 1E4L0170-0022, 1E4L0170-0025, 1E4L0170-0027, 1E4L0170-0031, 1E4L0170-0032, 1E4L0170-0033, 1E4L0170-0045, 1E4L0170-0047, 1E4L0170-0049, 1E4L0170-0050, 1E4L0170-0052, 1E4L0170-0053, 1E4L0170-0056, 1E4L0170-0058, 1E4L0170-0061, 1E4L0170-0066 and 1E4L0170-0067 are primary screening positive F1 mice.
[0058] Southern blot screening strategy for verifying F1 generation positive mice with A Probe-A and 5' Probe-A is shown in Figure 2, and the specific design is as follows (ScaI and Eco53KI are shown as Southern blot enzyme cutting sites):
[0059] ScaI and Eco53KI are used as Southern blot enzyme cutting sites. 5' Probe-A is used to detect whether correct recombination occurs. If correct recombination occurs, two bands of wild type and mutant will appear. A Probe-A is used to detect whether random insertion occurs. If there is no random insertion, one band of mutant will appear. Tail DNA of 14 F1 generation mice that are positive in PCR preliminary screening is extracted for Southern blot detection, and the results are shown in Figure 11.
[0060] Southern blot detection shows that 1E4L0170-0056, 1E4L0170-0058, 1E4L0170-0061, 1E4L0170-0066 and 1E4L0170-0067 are correct recombination and have no random insertion.
[0061] After PCR, Southern blot and sequencing verification, finally 5 F1 generation positive mice are obtained, and the specific information is as follows (5 F1 generation positive mice obtained after PCR, Southern blot and sequencing verification are shown):
[0062] It is shown that the FKBP8 humanized knock-in mouse model is successfully constructed in the embodiment.
[0063] The above examples only illustrate the present application and do not constitute a limitation on the protection scope of the present application. Any design similar to the present application is within the protection scope of the present application.
Claims
1. A method for constructing a humanized FKBP8 knock-in animal model, characterized in that, The method comprises the following steps: (1) constructing a targeting vector for humanized FKBP8 gene knock-in, and transfecting the targeting vector, Cas9 and sgRNA into a fertilized egg of a donor animal; (2) transplanting the surviving fertilized egg cell into the oviduct of a pseudopregnant animal to obtain an F0 generation animal, identifying the F0 generation animal, and the animal showing positive homologous recombination is a chimera animal; (3) mating the chimera animal with a wild-type animal to obtain an F1 generation animal and identifying the F1 generation animal, and the F1 animal showing positive homologous recombination is a humanized FKBP8 gene knock-in animal; wherein: the step (1) is specifically as follows: designing a targeting strategy based on the EGE system developed by CRISPR / Cas9, replacing the extracellular region of the FKBP8 gene of the donor animal with the extracellular region of the FKBP8 gene of the human, designing sgRNAs in the Exon2 and Exon8 of the FKBP8 gene of the donor animal respectively, designing sgRNAs in the 5' end and 3' end target regions, detecting in-vitro activity, selecting sgRNAs in combination with the specificity and cleavage position of sgRNA, carrying out in-vitro transcription on the sgRNAs and Cas9, and preparing RNA for microinjection; constructing a recombinant targeting vector according to the targeting scheme, and transfecting the recombinant targeting vector, Cas9 and sgRNAs into a fertilized egg of a donor animal; in the step (1), four sgRNAs are designed in the 5' end target region, and the sequences are shown as SEQ ID NO. 1-4; four sgRNAs are designed in the 3' end target region, and the sequences are shown as SEQ ID NO. 5-8; the 5' target site and the 3' target site with the highest activity are selected for the next experiment; in the step (2), the genotype of the F0 generation animal is identified by PCR; the primer sequences are shown as SEQ ID NO. 9-12.
2. The method for constructing a humanized FKBP8 gene knock-in animal model as described in claim 1, characterized in that: in the step (1), the sequence of the 5' target site EGE-HJL-0170-A-sgRNA3 with the highest activity is shown as SEQ ID NO. 3; and the sequence of the 3' target site EGE-HJL-0170-A-sgRNA7 with the highest activity is shown as SEQ ID NO.
7.
3. The method for constructing a humanized FKBP8 knockin animal model according to claim 2, wherein the human FKBP8 gene is inserted into the genome of the animal model at the same position as the mouse FKBP8 gene. The step (1) is detected by UCA TM in vitro activity of sgRNA.
4. The method for constructing a humanized FKBP8 knockin animal model according to claim 1, wherein the human FKBP8 gene is inserted into the genome of the animal model at the same position as the mouse FKBP8 gene. in the step (3), the F1 generation animal is identified by PCR and Southern blot; the DNA of the F1 generation animal identified as positive by PCR is detected by Southern blot and sequencing, correct recombination is confirmed, and there is no random insertion, and the F1 generation humanized FKBP8 gene knock-in animal model is successfully constructed.
5. The method for constructing a humanized FKBP8 knockin animal model according to claim 1, wherein the human FKBP8 gene is inserted into the genome of the animal model at the same position as the mouse FKBP8 gene. The animal is a mouse.
6. The humanized FKBP8 gene knock-in animal model constructed by the method according to any one of claims 1-5.
7. The humanized FKBP8 gene knock-in animal model according to claim 6 is used in screening drugs targeting FKBP8 gene and its products or researching the related physiological and pathological mechanisms of FKBP8 gene and its products.