Cartilage tissue-specific Cul7 gene knockout mouse model and method and application thereof established based on the Cre / LoxP system

Abstract

The present invention relates to a specific Cul7 gene knockout mouse model and a method and application for establishing the same based on the Cre / LoxP system. Specifically, parental Cul7<supgt;fl / fl< / supgt; mice are crossed with Col2a1-CreERT2 mice to obtain offspring with the genotype Cul7<supgt;fl / +< / supgt>; Col2a1-CreERT2 mice. The obtained Cul7<supgt;fl / +< / supgt>; Col2a1-CreERT2 mice are then crossed with Cul7<supgt;fl / fl< / supgt> mice, and Cul7<supgt;fl / fl< / supgt>; Col2a1-CreERT2 mice are obtained through genotyping of the offspring mice. The Cul7<supgt;fl / fl< / supgt>; Col2a1-CreERT2 mice are intraperitoneally injected with tamoxifen after birth, and a Cul7 cKO mouse model is successfully constructed, which shows obvious growth retardation and short limbs, consistent with the clinical manifestations of patients with 3M syndrome. The successful establishment of the model in the present invention provides a reliable experimental model for further studying the Cul7 gene signaling pathway and the pathogenic mechanism of 3M syndrome in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a cartilage tissue-specific Cul7 gene knockout mouse model, a method for establishing the same based on the Cre / LoxP system, and an application thereof. Background Art

[0002] 3M syndrome (OMIM 273750, 612921, 614205) is a rare autosomal recessive genetic disorder of skeletal dysplasia. Its main clinical manifestations are severe intrauterine and postnatal growth retardation, skeletal dysplasia, significant short stature (< -4 SD), accompanied by facial deformities, but normal intelligence level and endocrine function. So far, the molecular mechanism related to the pathogenesis of 3M syndrome is not fully understood. Currently, three pathogenic gene mutations have been discovered, including Cul7, OBSL1, and CCDC8, among which Cul7 accounts for about 70% and is the most common form of mutation.

[0003] 3M syndrome is characterized by significant short stature. The linear growth of long bones depends on the proliferation, differentiation, and apoptosis of growth plate chondrocytes. Cul7 is expressed in the cytoplasm, nucleus, and cell membrane of chondrocytes in each zone of the mouse growth plate, and with the increase of the week age, the expression level decreases most significantly in the proliferative zone. In addition, Huber et al. found in their research that in the femoral growth plate of fetuses with 3M syndrome caused by Cul7 gene mutation, the density and volume of chondrocytes in the resting zone and proliferative zone increase. These studies all indicate that Cul7 is involved in the growth and proliferation of growth plate chondrocytes.

[0004] Cul7 (p185, KIAA0076) was first discovered by Nomura N et al. in 1994. Its encoding gene is located on the short arm of human chromosome 6 (6p21.1) and is expressed in almost all tissues of the human body. Cul7 belongs to a member of the Cullin family and plays an important role in signal transduction, cycle regulation, apoptosis, and senescence. Cul7 is also closely related to the occurrence of tumors. It is expressed in a variety of malignant tumors, and multiple studies have shown that it can inhibit tumor cell apoptosis and promote tumor cell proliferation and growth.

[0005] Previous studies have shown that mice with global Cul7 gene knockout exhibit severe intrauterine growth retardation and are prone to death due to respiratory distress during the perinatal period, which hinders further research on postnatal growth and development. Constructing spontaneous animal disease models by specifically knocking out disease-related target genes to cause the deletion of specific proteins or cytokines has now been increasingly recognized. The application of the Cre / LoxP system can achieve specific knockout of target genes in specific types of tissues or organs, avoiding the embryonic lethality problems caused by the knockout of some genes related to growth and development, and has obvious advantages in the construction of human animal disease models, and there have been successful reports in many literatures.

[0006] In view of this, in order to further study the pathogenic mechanism of 3M syndrome, it is necessary to construct a cartilage tissue-specific Cul7 gene conditional knockout mouse model, and there is currently no report on the establishment of a cartilage tissue-specific Cul7 gene conditional knockout mouse model based on the Cre / LoxP system. Summary of the Invention

[0007] The purpose of the present invention is to overcome the defects existing in the prior art, provide a cartilage tissue-specific Cul7 gene conditional knockout mouse model, and at the same time provide a method for establishing a cartilage tissue-specific Cul7 gene conditional knockout mouse model based on the Cre / LoxP system, providing a research basis for further studying the pathogenic mechanism of 3M syndrome and other diseases related to the Cul7 gene.

[0008] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0009] The present invention provides a method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system, which includes the following steps:

[0010] S1: Select parental Cul7 fl / fl mice and Col2a1-CreERT2 mice for hybridization to obtain the genotype Cul7 fl / + ; Col2a1-CreERT2 mice;

[0011] S2: Hybridize the obtained genotype Cul7 fl / + ; Col2a1-CreERT2 mice with the genotype Cul7 fl / fl mice, and obtain Cul7 fl / fl ; Col2a1-CreERT2 mice through the genotyping of the offspring mice.

[0012] S3: Cul7 fl / fl; At postnatal day 11, Col2a1-CreERT2 mice were intraperitoneally injected with tamoxifen to establish a Cul7 cKO mouse model.

[0013] As some preferred embodiments of the present invention, the gene identification in step S2 is to amplify gene fragments by PCR according to the position of the Flox region during the construction of the targeting vector, and determine the genotype of the mice by agarose gel electrophoresis and according to the length of the gene fragments and the presence or absence of products.

[0014] As some preferred embodiments of the present invention, in the PCR reaction system, the sequences of the Cul7 primer pairs are as follows:

[0015] Forward P1, 5’-TCCCAGAAACTTACGATGTGC-3’;

[0016] Reverse P2, 5’-CTTGGGGGTGGGGAGAGTG-3’;

[0017] The sequences of the Cre transgenic primer pairs are as follows:

[0018] Forward P3, 5’-TCGATGCAACGAGTGATGAG-3’;

[0019] Reverse P4, 5’-TCCATGAGTGAACGAACCTG-3’.

[0020] As some preferred embodiments of the present invention, the verification of the Cul7 cKO mouse model in step S3 is to amplify gene fragments by PCR according to the position of the entire Flox region during the construction of the targeting vector, and determine the genotype of the mice by agarose gel electrophoresis or Sanger sequencing.

[0021] As some preferred embodiments of the present invention, the sequences of the primer pairs for the entire Flox region are as follows:

[0022] Forward P5, 5’-GATGAGGGCCAGTTCCGACAGA-3’;

[0023] Reverse P6, 5’-TGGGCAGCAAAGGCAGACG-3’.

[0024] As some preferred embodiments of the present invention, in step S3, the injection dose of tamoxifen is 75 mg / kg, and the injection time is 3 days, which can not only ensure the knockout efficiency but also ensure the survival rate of the mice.

[0025] On the other hand, the present invention provides a cartilage tissue-specific Cul7 gene knockout mouse model established by the above method.

[0026] On the other hand, the present invention provides the application of the cartilage tissue-specific Cul7 gene knockout mouse model established by the above method in the research on the Cul7 gene signal transduction pathway and the pathogenic mechanism of 3M syndrome, as well as in drug screening.

[0027] The beneficial effects produced by adopting the above technical solutions are as follows:

[0028] The present invention has successfully established a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system for the first time, which shows obvious growth and development retardation and short limbs, consistent with the clinical manifestations of patients with 3M syndrome.

[0029] The successful establishment of the model of the present invention provides a reliable experimental model for further studying the Cul7 gene signal transduction pathway and the pathogenic mechanism of 3M syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art.

[0031] Figure 1 For the construction idea diagram of the Cul7 flox / flox mouse model of the present invention;

[0032] Figure 2 For the experimental flow chart of Example 1 of the present invention;

[0033] Figure 3 For the genotype verification diagram of the Cul7 cKO gene mouse model;

[0034] Wherein: (A) Cul7 fl / fl Homozygous has a single band of 399 bp; Cul7 fl / + Heterozygous has two bands of 399 bp and 258 bp, Cul7 + / + Wild type has a single band of 258 bp; Cre recombinase has a single band of 400 bp; (B) Cul7 cKO has a single band at 903 bp; Cre has not played a role and has a single band at 2484 bp;

[0035] Figure 4 For the Sanger sequencing diagram of the Cul7 cKO gene mouse model of the present invention;

[0036] Figure 5 For the Western blot diagram of the Cul7 cKO gene mouse model of the present invention;

[0037] Figure 6This is the comparison graph of Cul7 protein expression in Example 2 of the present invention;

[0038] Figure 7 This is the immunohistochemical staining graph of Cul7 in Example 2 of the present invention;

[0039] Figure 8 This is the appearance morphology photo of the Cul7 cKO gene mouse model and the control group mice in Example 2 of the present invention;

[0040] Among them: (A) The appearance photos of the two groups of mice; (B) The morphological photos of the X-ray films; (C) The appearance morphology photos of the hind limbs of the two groups of mice;

[0041] Figure 9 This is the comparison graph of the nose-to-tail length of the Cul7 cKO gene mouse model and the control group mice in Example 2 of the present invention;

[0042] Figure 10 This is the comparison graph of the femur length of the Cul7 cKO gene mouse model and the control group mice in Example 2 of the present invention;

[0043] Figure 11 This is the comparison graph of the tibia length of the Cul7 cKO gene mouse model and the control group mice in Example 2 of the present invention. Detailed implementation manners

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0045] The experimental animals used in this part are sourced as follows:

[0046] In this part, parental Cul7 flox / flox mice with a C57BL / 6 genetic background and Col2a1-CreERT2 mice, SPF grade, half male and half female, 8 weeks old, with an average weight of about 20 g, were purchased from Shanghai Model Organisms Center, Inc., production license number: SCXK (Shanghai) 2017-0010; they were raised in a constant temperature and humidity SPF-grade environment in the Animal Experiment Center of the Second Hospital of Hebei Medical University, and the use license number: SYXK (Hebei) 2021-003; all animal procedures and protocols were approved by the Ethics Committee of the Second Hospital of Hebei Medical University (Hebei, China) (approval number: 2022-AE252).

[0047] In this section, an innovative cartilage tissue-specific knockout mouse model was established to replace the systemic Cul7 gene knockout, providing a reliable experimental model for the pathogenic mechanism of 3M syndrome. 3M syndrome is characterized by obvious short stature. Cartilage is an important and special supporting organ and tissue in animals. During embryonic development, cartilage forms first and dominates the osteogenic process during development and finally forms mature bones (endochondral ossification). Endochondral ossification is the main formation method of bones such as long bones, including most of the major limb bones of the whole body such as the femur, tibia, ulna, radius, humerus, etc., as well as the main trunk supporting bones such as vertebrae and ribs. Their generation and development all originate from the endochondral ossification process.

[0048] In this section, the Cre / LoxP system was used to establish a conditional gene knockout mouse model of Cul7. Two kinds of transgenic animals were needed. One was the Cul7 transgenic mouse with LoxP sequences introduced into the gene sequence of the targeted knockout group, and the other was the CreERT2 transgenic mouse driven by the Col2a1 promoter. Then, conditional gene knockout mice containing both the Cre recombinase gene and LoxP sequences in vivo were constructed by hybridization.

[0049] 1. Cul7 flox / flox The construction idea of the mouse model was as follows:

[0050] (1) Name of the target gene (Ensembl ID): Cul7 (ENSMUSG00000038545)

[0051] (2) Ensembl website link of the target gene:

[0052]

[0053] (3) Transcript targeted (Ensembl ID): Cul7-201 (ENSMUST00000043464.13)

[0054] (4) Exons targeted by Flox: exon 5-7. Exon 567 of CUL7 plays an important role. Therefore, LoxP sequences were added on both sides of exon 567, as shown in Figure 1 .

[0055] 2. Col2a1-CreERT2 mice

[0056] (1) Cre-ERT2 mice are a type of mice that express a fusion protein of a ligand-binding domain mutant (ERT) of the estrogen receptor (ER) and Cre recombinase. As one of the main load-bearing tissue organs of collagen, cartilage expresses various collagens including col1a1, col2a1, and col10a1. Among them, col2a1 maintains extremely high expression throughout the entire period of chondrocyte formation and development, becoming one of the specific markers of chondrocytes and cartilage tissue. Therefore, in this study, CreERT2 transgenic mice driven by the Col2a1 promoter were used to specifically regulate the expression of Cre in cartilage tissue.

[0057] (2) In the absence of tamoxifen (TAM, Tamoxifen) induction, Cre-ERT2 is in an inactive state in the cytoplasm; when induced by Tamoxifen, the metabolite 4-OHT (an estrogen analogue) of Tamoxifen binds to ERT, enabling Cre-ERT2 to enter the nucleus and exert Cre recombinase activity, specifically recognizing the LoxP sequence. When two LoxP sequences are located on the same DNA strand and in the same direction, the DNA fragment between the two LoxP sequences is knocked out.

[0058] The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system is described in detail below in combination with specific embodiments.

[0059] Example 1 Construction of Cul7 cKO mouse model

[0060] 1.1 Breeding and reproduction of mice

[0061] The parental Cul7 fl / fl mice were crossed with Col2a1-CreERT2 mice to obtain offspring with the genotype Cul7 fl / + ; the Col2a1-CreERT2 mice were then crossed with mice with the genotype Cul7 fl / fl to obtain newly born mice. During the breeding process of the model mice, male and female mice were caged and mated at a ratio of 1:2 or 1:3. By genotyping the offspring mice, Cul7 fl / fl ; Col2a1-CreERT2 mice were obtained. The schematic diagram of the experimental procedure is shown in Figure 2 .

[0062] 1.2 Genotyping of mouse offspring

[0063] According to the position of the Flox region during the construction of the targeting vector, gene fragments were amplified by PCR, and agarose gel electrophoresis was used to determine the genotype of the mice based on the length of the gene fragments and the presence or absence of the products.

[0064] 1.2.1 DNA Extraction

[0065] Cut the toes of 5-day-old mice after birth and place them in 2 ml EP tubes. Add 230 µl Buffer GA and 20 µl Proteinase K to each tube, and incubate overnight at 55 °C in a water bath until the tissue is completely enzymatically digested; add 250 µl Buffer GB, vortex thoroughly, and incubate at 70 °C in a water bath for 10 min. Purify and elute the genome.

[0066] 1.2.3 Amplification and Agarose Gel Electrophoresis of Mouse Toe Cre and Cul7 Genes

[0067] The total volume of the PCR reaction system is 50 μL. The primer sequences for Cul7 are as follows:

[0068] Forward P1, 5’-TCCCAGAAACTTACGATGTGC-3’;

[0069] Reverse P2, 5’-CTTGGGGGTGGGGAGAGTG-3’;

[0070] The primer sequences for Cre transgenic:

[0071] Forward P3, 5’-TCGATGCAACGAGTGATGAG-3’;

[0072] Reverse P4, 5’-TCCATGAGTGAACGAACCTG-3’.

[0073] 2 μL of the forward primer (10 μmol / L), 2 μL of the reverse primer (10 μmol / L), 1 - 2 μL of the DNA stock solution (depending on the concentration, the usage amount is 10 - 400 ng), 25 μL of MonAmp™ 2× Taq Mix Pro (+Dye), 19 - 20 μL of ddH2O. PCR (Type: 5020, Brand: Thermo Fisher) amplification conditions: 95 °C for 10 min; 95 °C for 30 s, 62 °C for 30 s, 72 °C for 20 s, for 32 cycles; 72 °C for 5 min.

[0074] Dissolve 1.4 g of agarose in 70 mL of 1×TAE (Tris-acetate) to prepare a 2% agarose solution. After boiling the gel, preparing the plate, and pouring the gel, take 10 μL of the amplified product for loading, and add 6 μL of DNA marker to the empty slot at the right end of the gel plate. After electrophoresis, observe with a gel imaging system (Model: Universal Hood Ⅲ, Brand: BioRad). Electrophoresis conditions: 120 V, 30 min.

[0075] 1.3 Construction of Cul7 cKO mouse model

[0076] For Cul7 fl / fl ; Col2a1-CreERT2 mice were intraperitoneally injected with tamoxifen (dissolved in corn oil, 20 mg / ml) at a dose of 75 mg / kg starting from the 11th day after birth, once a day for 3 consecutive days. After knockout of the mice, genotype verification was performed (the method was the same as 1.2). The primer sequences for the entire Flox region were as follows:

[0077] Forward P5, 5’-GATGAGGGCCAGTTCCGACAGA-3’;

[0078] Reverse P6, 5’-TGGGCAGCAAAGGCAGACG-3’.

[0079] The amplified products were subjected to Sanger sequencing: the PCR products were purified, quantified, sequenced on the machine, and the sequence analysis results were obtained.

[0080] Example 2 Verification of mouse model

[0081] 2.1 Verification of mouse model at gene level - agarose gel electrophoresis and Sanger sequencing

[0082] Conditional gene knockout of Cul7 was screened out by agarose gel electrophoresis identification results fl / fl ; Col2a1-CreERT2 gene mice. For Cul7 fl / fl homozygotes had a single band at 399 bp, and Cre recombinase had a single band at 400 bp ( Figure 3 A). After injection of tamoxifen, Cre exerted its effect and there was a single band at 903 bp, obtaining the Cul7 cKO mouse model, as shown in Figure 3 B. The Sanger sequencing results confirmed the PCR results again, as shown in Figure 4 , that is, the Cul7 gene was conditionally knocked out in cartilage, verifying the successful construction of the Cul7 cKO mouse model.

[0083] As Figure 4 shown, marked as the excised fragment of Cul7.

[0084] 2.2 Verification of mouse model at protein level - Western blot and immunohistochemistry

[0085] Western blotting showed that the expression level of Cul7 protein in the experimental group at 3 weeks was significantly decreased compared with the control group ( Figure 5 ), and their expression levels were normalized to the expression level of GAPDH (the difference was statistically significant, t= 27.956, P< 0.001 )( Figure 6 ). The results of Cul7 immunohistochemical staining were consistent with the above results ( Figure 7 ), further verifying the knockout efficiency of tamoxifen.

[0086] 2.3 Verification of the mouse model at the macroscopic morphological level - Observation of the external appearance and X-ray of mice

[0087] Comparing the gross external appearance and X-ray films of mice at the 4th week, it was found that the Cul7 gene knockout mice in the experimental group showed obvious limb shortening compared with the control group mice, manifested as short and deformed limbs, and the abdomen was slightly bulged, as shown in Figure 8 ; Comparing the external appearance of the hind limbs of the two groups of mice after dissection, the experimental group showed: slender tubular bones, as shown in Figure 8 C; X-ray observation showed that the limb length of the experimental group mice was significantly shortened from 3 to 6 weeks, and the nose-tail length, tibia length, and femur length were all shorter than those of the control group, and the differences were statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). The length data comparison of the nose-tail length, tibia length, and femur length of the two groups is shown in Figures 9 - 11 .

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system, characterized in that, It includes the following steps: S1: Select parental Cul7 fl / fl Mice were crossed with Col2a1-CreERT2 mice to obtain the genotype Cul7 fl / + ; Col2a1-CreERT2 mice; S2: The obtained genotype Cul7 fl / + ; Col2a1-CreERT2 mice were crossed with genotype Cul7 fl / fl mice, and Cul7 fl / fl ; Col2a1-CreERT2 mice were obtained by genotyping the offspring mice; S3: Add Cul7 fl / fl ; For Col2a1-CreERT2 mice, tamoxifen was intraperitoneally injected starting from the 11th day after birth to establish a Cul7 cKO mouse model.

2. The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system according to claim 1, characterized in that, The gene identification in step S2 is to use the position of the Flox region during the construction of the targeting vector to amplify gene fragments by PCR, and determine the genotype of the mouse by agarose gel electrophoresis and according to the length of the gene fragment and the presence or absence of the product.

3. The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system according to claim 2, characterized in that, In the PCR reaction system, the sequences of the Cul7 primer pairs are as follows: Forward P1, 5’-TCCCAGAAACTTACGATGTGC-3’; Reverse P2, 5’-CTTGGGGGTGGGGAGAGTG-3’; The sequences of the Cre transgenic primer pairs are as follows: Forward P3, 5’-TCGATGCAACGAGTGATGAG-3’; Reverse P4, 5’-TCCATGAGTGAACGAACCTG-3’.

4. The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system according to claim 1, characterized in that, The verification of the Cul7 cKO mouse model in step S3 is to use the position of the entire Flox region during the construction of the targeting vector to amplify gene fragments by PCR, and determine the genotype of the mouse by agarose gel electrophoresis or Sanger sequencing.

5. The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system according to claim 4, characterized in that, The sequences of the primer pairs for the entire Flox region are as follows: Forward P5, 5’-GATGAGGGCCAGTTCCGACAGA-3’; Reverse P6, 5’-TGGGCAGCAAAGGCAGACG-3’.

6. The method for establishing a cartilage tissue-specific Cul7 gene knockout mouse model based on the Cre / LoxP system according to claim 1, characterized in that, In step S3, the injection dose of tamoxifen is 75 mg / kg, and the injection time is 3 days.

7. A cartilage tissue-specific Cul7 gene knockout mouse model established by the method according to any one of claims 1-5.

8. Application of the cartilage tissue-specific Cul7 gene knockout mouse model according to claim 7 in the research on the Cul7 gene signal transduction pathway and the pathogenic mechanism of 3M syndrome and drug screening.

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