A method for constructing an animal model of ciliopathy and its application

Abstract

The present application relates to the field of biotechnology, and specifically discloses a method for constructing an animal model of ciliopathy and its application. The present application uses the CRISPR / Cas9 system to perform gene editing on the CEP44 gene, and obtains a mouse model with a CEP44 pathogenic gene knockout. The CEP44 pathogenic gene knockout mouse model is very similar to the symptoms of human ciliopathy, and can be used not only to study the molecular mechanism of ciliary growth and the pathogenesis of ciliopathy, but also for clinical diagnosis and treatment and the screening of related therapeutic drugs.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for constructing a ciliary disease animal model and its application. Background Art

[0002] Ciliary disease is a disease caused by defects in the structure and function of cilia. Although ciliary disease is generally considered a rare disease, due to the fact that there are dozens of types of ciliary diseases, its incidence in the population is similar to the well-known Down syndrome, reaching 1:1000. Ciliary diseases generally have clinical symptoms involving the development of multiple organs, such as polydactyly, skeletal dysplasia, mental retardation, and cardiovascular and cerebrovascular dysfunction. The complexity of the manifestations of ciliary diseases has caused great difficulties in clinical diagnosis and treatment. At present, there is no effective cure for ciliary diseases in clinical practice. Therefore, it is necessary to further clarify the pathogenic mechanism of ciliary diseases.

[0003] In 2009, Baker and Beales proposed that more than 72 syndromes may be ciliopathies. So far, more than 30 different types of ciliopathies have been discovered. However, the pathogenic genes of some ciliopathies cannot be confirmed. In addition, some suspected but unconfirmed ciliopathies, such as Walker-Warburg (WWS) syndrome, have not yet found mutations in cilia-related genes. Therefore, many pathogenic genes of ciliopathies have not yet been discovered. Finding new pathogenic genes of ciliopathies is of great significance for a deeper understanding of the regulatory mechanism of cilia growth and the diagnosis and treatment of ciliopathies. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a method for constructing a ciliary disease animal model and its application.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides the use of the CEP44 pathogenic gene in preparing an animal model for diagnosing ciliopathy, and the accession number of the CEP44 pathogenic gene is: NM_001040157.3.

[0007] This application found that knocking out the Cep44 gene impaired the growth of cilia in mice, and found that the number of cilia in CEP44 knocked out MEF cells decreased significantly. A similar phenomenon was also found when knocking down the CEP44 gene in hTERT-RPE1 cells. This application discovered a possible new pathogenic gene for ciliopathy, which is of great significance for in-depth understanding of the regulatory mechanism of ciliary growth and the diagnosis and treatment of ciliopathy.

[0008] The present application provides a method for constructing an animal model of ciliopathy, wherein the animal model of ciliopathy is obtained by knocking out the CEP44 pathogenic gene.

[0009] As a preferred embodiment of the method for constructing the ciliopathy animal model described in the present application, the ciliopathy animal model is obtained by knocking out exons 3 to 6 of the CEP44 pathogenic gene.

[0010] As a preferred embodiment of the method for constructing the ciliopathy animal model described in the present application, the following steps are included:

[0011] 1) respectively transcribing the gRNA1 having a nucleotide sequence as shown in SEQ ID NO: 1 and the gRNA2 having a nucleotide sequence as shown in SEQ ID NO: 2 into mRNA in vitro to obtain active gRNA1 and gRNA2;

[0012] 2) microinjecting the gRNA1, gRNA2, and Cas9 protein into mouse fertilized eggs to obtain F0 generation mice;

[0013] 3) The F0 generation mice are mated with wild-type mice, and the F1 generation heterozygous mice are selected from the genotype identification results of the F1 generation mice. The heterozygous mice are mated to select homozygotes to obtain a mouse model with CEP44 pathogenic gene knockout, that is, a ciliopathy animal model.

[0014] The present application discloses the use of the CRISPR / Cas9 system to perform gene editing on the CEP44 gene to obtain the ciliopathy animal model of the present application. The ciliopathy animal model obtained in the present application is very similar to the symptoms of human ciliopathy, and can not only be used to study the molecular mechanism of ciliary growth and the occurrence mechanism of ciliopathy.

[0015] Among them, CRISPR / Cas9 is a technology guided by sgRNA and using Cas9 nuclease to edit targeted genes. Its working principle is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA / crRNA complex. This complex will guide the Cas9 protein to cut double-stranded DNA at the sequence target site paired with crRNA. By artificially designing two RNAs, crRNA and tracrRNA, they are transformed into sgRNA with guiding function, thereby guiding the Cas9 protein to cut DNA at a fixed point and produce a double-stranded DNA gap with a flat end, thereby initiating the DNA damage repair mechanism, mainly connecting the sequences at the upstream and downstream ends of the break through non-homologous end joining (Non-homologous end joining, NHEJ) or homologous recombination (Homologous recombination, HR).

[0016] As a preferred embodiment of the method for constructing a ciliopathy animal model described in the present application, the step 3) comprises the following steps:

[0017] According to exons 3 to 6 of the CEP44 pathogenic gene, PCR amplification was performed on F0 positive mice and wild-type mice using a primer set with sequences as shown in SEQ ID NOs: 3 to 6.

[0018] As a preferred embodiment of the method for constructing a ciliary disease animal model described in the present application, the concentrations of gRNA1 and gRNA2 are both 100 ng / μl.

[0019] The present application also provides a ciliopathy animal model prepared by the construction method of the above-mentioned ciliopathy animal model.

[0020] The mouse CEP44 exons 3-6 were knocked out using CRISPR / Cas9 technology to obtain a mouse model with CEP44 pathogenic gene knockout. PCR results showed that the target fragment of the mouse model with CEP44 pathogenic gene knockout was knocked out.

[0021] In order to further verify the knockout efficiency of the CEP44 pathogenic gene knockout mouse model, the embryonic proteins of wild-type mice and CEP44 pathogenic gene knockout mouse models were extracted, and the protein levels of knockout mice were detected by immunoblotting experiments. The results showed that CEP44 protein existed in wild-type mice, and this band was missing in the CEP44 pathogenic gene knockout mouse model, confirming that the CEP44 pathogenic gene knockout mouse model was successfully prepared. Further analysis found that CEP44 knockout mice were embryonic lethal and showed ciliopathy-related phenotypes such as brain malformations, polydactyly, and polycystic kidneys.

[0022] In addition, the mouse model with CEP44 pathogenic gene knockout obtained in the present application has abnormal skull, missing basesphenoid, occipital and anterior sphenoid bones, and cleft palate in the mouse. It was also observed that the mouse model with CEP44 pathogenic gene knockout had short limbs and polydactyly. Further analysis revealed that the sternum and vertebrae of the mouse model with CEP44 pathogenic gene knockout were misaligned and structurally abnormal.

[0023] The present application also provides a gRNA for constructing a mouse model of CEP44 pathogenic gene knockout, wherein the gRNA includes a gRNA1 with a nucleotide sequence as shown in SEQ ID NO: 1 and a gRNA2 with a nucleotide sequence as shown in SEQ ID NO: 2.

[0024] The present application also provides the use of a ciliopathy animal model in screening drugs for treating ciliopathy.

[0025] This application found that Cep44 knockout mice showed characteristics of cilia-related diseases, including perinatal death, brain malformation, polydactyly, polycystic kidney and skeletal dysplasia. Further analysis found that Cep44 knockout mice had impaired cilia growth. The number of cilia was significantly reduced in CEP44 knockout MEF cells, and a similar phenomenon was found in hTERT-RPE1 cells by knocking down CEP44. This application discovered a possible new pathogenic gene for cilia disease, which is of great significance for in-depth understanding of the regulatory mechanism of cilia growth and the diagnosis and treatment of cilia disease.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The present application provides a method for constructing an animal model of ciliopathy and its application, and also discovers a possible new pathogenic gene CEP44 for ciliopathy. The present application uses the CRISPR / Cas9 system to perform gene editing on the CEP44 gene, and obtains a mouse model with the CEP44 pathogenic gene knocked out. The CEP44 pathogenic gene knocked out mouse model has symptoms very similar to those of human ciliopathy, and can be used not only to study the molecular mechanism of ciliary growth and the pathogenesis of ciliopathy, but also for clinical diagnosis and treatment and the screening of related therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The mouse model with CEP44 pathogenic gene knockout shows the characteristics of cilia-related diseases (including perinatal death, brain malformation, polydactyly and polycystic kidney) (A is the knockout of Cep44 gene using CRISPR / Cas9 technology, and the 3rd to 6th exons of the transcript are knocked out. B is the genotype identification of CEP44 wild-type, heterozygous and homozygous mice using PCR. C is the tissue lysate (from Cep44) using antibodies against Cep44 and tubulin+ / + Wild-type mice and Cep44 - / - Extracts from embryonic limbs) were subjected to immunoblot analysis. D is Cep44 + / + Wild-type mice and Cep44 - / - Whole embryo image of homozygous knockout mouse embryo at E18.5, Cep44 - / - Homozygous knockout mice are small, have extra fingers, abnormal head development, and systemic bleeding. E stands for Cep44 + / + Wild-type mice and Cep44 - / - The number of fingers / toes of homozygous knockout mice at E18.5. F is Cep44 + / + Wild-type mice and Cep44 - / - Hematoxylin and eosin (H&E) staining of coronal sections of the head of homozygous knockout mice at E14.5 days. G is Cep44 + / + Wild-type mice and Cep44 - / - Coronal section of embryonic kidney of homozygous knockout mice at E18.5 days, stained with hematoxylin-eosin. The enlarged part of the dotted box is the glomerulus. H is Cep44 + / + Wild-type mice and Cep44 - / - The formation of cilia in renal tubular epithelial cells of homozygous knockout mice at E18.5 days. The dotted line indicates the renal tubules; I indicates wild type and Cep44 - / - Kidney images of homozygous knockout mice at E18.5, Cep44 - / - The kidneys of homozygous knockout mice are abnormal in appearance.)

[0029] Figure 2 This is a staining image of the mouse model of CEP44 pathogenic gene knockout using Alizarin Blue and Alizarin Red;

[0030] Figure 3 These are the immunofluorescence images and cilia count results of the CEP44 pathogenic gene knockout mouse model. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0033] Example 1: A method for constructing a ciliopathy animal model

[0034] This embodiment provides a method for constructing an animal model of ciliopathy, comprising the following steps:

[0035] 1. Production of transgenic mice

[0036] 1. sgRNA preparation:

[0037] (1) Design of sgRNA:

[0038] Two sgRNAs were designed based on the sequence of mouse CEP44 exons (exons 3-6), namely 5'-GCCTGGGCTCATGTCTCGGA-3' (gRNA1, SEQ ID NO: 1) and 5'-ATATTTTCCCCAAGGGGCAT-3' (gRNA2, SEQ ID NO: 2).

[0039] (2) Synthesis and purification of sgRNA transcription template:

[0040] PCR was used to synthesize the transcription template of sgRNA (sgRNA complementary fragment), and the PCR reaction system is shown in Table 1 below:

[0041] Table 1

[0042] sgRNA (forward) (concentration 10 μM) 2μl sgRNA (reverse) (concentration 10 μM) 2μl 2x Taq Master Mix (NoviZan, Cat. No. P112-01) 25μl Double distilled water 21μl Total volume 50μl

[0043] The PCR reaction program is shown in Table 2 below:

[0044] Table 2

[0045] temperature time Number of cycles 94℃ 2min 1 94℃ 30s 40 55℃ 30s 40 72℃ 30s 40 72℃ 5min 1 4℃ Always 1

[0046] After the PCR reaction is completed, the PCR product is subjected to agarose gel electrophoresis, and then purified using a PCR product purification kit. The concentration of the purified product is measured using an ELISA reader.

[0047] (3) In vitro transcription of sgRNA:

[0048] Set up the reaction system on ice as shown in Table 3:

[0049] Table 3

[0050]

[0051]

[0052] After preparing the reaction system on ice, incubate at 37°C for 2.5 hours. Then add 1 μl of DNase I to the reaction system and incubate at 37°C for 15 minutes. Then add 10 μl of ammonium acetate (5M) to the tube, mix well, and terminate the reaction.

[0053] (4) Purification of sgRNA:

[0054] Add 70 μL of water and 100 μL of phenol / chloroform / isoamyl alcohol to the reaction system prepared in step (3) in order, vortex to mix the liquid, and centrifuge at 12000 g for 1 minute. Transfer the upper aqueous phase to a new centrifuge tube, add 150 μL of chloroform, and use a vortex mixer to mix the liquid thoroughly. Centrifuge at 12000 g for 1 minute, transfer the upper aqueous phase to a new centrifuge tube again, and then add an equal volume of isopropanol for precipitation. After mixing, place at -20°C for 30 minutes. Then, centrifuge at 12000 g at 4°C for 15 minutes. Remove the supernatant, add 700 μL of 70% ethanol, and centrifuge at 12000 g at 4°C for 15 minutes. Discard the liquid, keep the precipitate and add 10 μL of pure water, and use an enzyme reader to measure the concentration. Dilute the sgRNA concentration to 100 ng / μl and store it in aliquots at -80°C.

[0055] 2. Preparation of vasectomized male mice:

[0056] (1) Anesthetize mice:

[0057] Grab the mouse and insert the injection needle into the subcutaneous part of the lower abdomen about 0.5 cm from the linea alba. The needle should not penetrate the mouse's abdominal cavity more than 1 cm. If the needle is inserted too shallowly, it is not easy to pierce the abdominal wall and it is easy to cause subcutaneous injection. If the needle is inserted too deep, it is easy to pierce the internal organs. When injecting, the mouse's head must be in a low position and the tail is slightly lifted to move the internal organs forward. After piercing the abdominal cavity, you can gently pull the syringe. If it is confirmed that no blood or intestinal contents flow into the abdominal cavity, it means that the needle has penetrated the abdominal cavity, and you can start to inject the medicine. You can pinch its tail to check whether the anesthesia is complete. Generally, the mouse is completely anesthetized in about 10 minutes.

[0058] (2) Vasectomy:

[0059] Place the mouse on a plate with the ventral surface facing up, fix the mouse's limbs on the plate with tape, and wipe the mouse's abdomen with 75% alcohol. Use scissors to cut 1 cm horizontally in the middle of the horizontal line of the upper end of the lower limb, and also cut the abdominal wall muscles. Use forceps to clamp one side of the fat pad downward to pull the testicle, epididymis and vas deferens out of the incision. Bluntly separate the vas deferens for about 1 cm, cut it on both sides with surgical scissors or burn it with red-hot forceps. Clamp the fat pad and put the testicle back into the abdominal cavity. Ligate the vas deferens on the other side in the same way as above, suture the abdominal wall, and suture the skin.

[0060] (3) After the operation, a cotton pad was laid to maintain the body temperature of the mouse. After waking up, the mouse was returned to the breeding room. Two weeks later, the mouse was mated with a female mouse. The thrombus was checked the next morning. The female mouse was checked to see if it was pregnant two weeks later to verify whether the ligation was successful.

[0061] 3. Preparation of pseudopregnant female ICR mice:

[0062] The night before fertilized egg transplantation, female ICR mice were mated with vasectomized male ICR mice (1 male per cage with 2-3 females, 5 cages in total). The next morning, the plugs were checked and plug-positive mice were selected and caged for later use.

[0063] 4. Superovulation of mouse eggs:

[0064] (1) At 12:00-1:00 pm, take a tube of 50 μl PMSG and dilute it to 1000 μl with saline. Inject 100 μl into the peritoneum of 5-6 week old C57BL / 6 female mice for a total of 10 mice.

[0065] (2) 44-48 hours after PMSG injection, take a 50 μl tube of hCG and dilute it to 1000 μl with saline. Inject hCG intraperitoneally, 100 μl per mouse. After hCG injection, mate each (or two) female C57 mice with a male C57 mouse in the same cage.

[0066] (3) At 9 a.m. the next day, use sharp forceps to check the plugs of C57 and ICR female mice after mating. The presence of white lumps at the vaginal opening of female mice indicates successful mating. Select C57 mice with plugs for egg retrieval.

[0067] 5. Collection of mouse fertilized eggs:

[0068] The mice were killed by dislocating their necks. A V-shaped opening was made on the abdomen of the mice with scissors. The fur on both sides of the V-shaped opening was pulled apart to expose the epithelium, or the abdominal skin was torn open by hand. The epithelium was cut open, and the uterus and ovaries were found on both sides of the internal organs. The connection between the uterus and ovaries was the fallopian tube, which was cut off and placed in M2.

[0069] Under the dissecting microscope, find the expansion ampulla of fallopian tube, tear open the ovum with fine pointed forceps and see that the ovum with cumulus cell flows out voluntarily. Subsequently, the fertilized egg with cumulus cell is moved in the M2 culture fluid containing hyaluronidase, repeatedly blow and suck several times and make it disperse, dissolve the cumulus cell and immediately use the straw drawn in advance to draw the ovum in the fresh M2 culture fluid and wash, and then the fertilized egg is drawn in the other fresh M2 culture fluid and carried out 3 times (discarding dead fertilized egg in transfer), until there is no tissue fragment and hemocyte in the visual field. At last, the fertilized egg is drawn in the M16 culture fluid drop of CO2 balance, and the drop is covered with a layer of embryonic grade mineral oil again, and put into 37 degree 5%CO2 incubator culture.

[0070] The dissection instruments, glassware, solutions, etc. used in the entire experimental process must be sterilized to prevent contamination. After the egg pronucleus develops clearly and fully, Cas9 protein (commercially purchased, product number Z00389, genscript) and sgRNA can be microinjected. Both Cas9 protein and sgRNA are diluted to 200ng / μl, and then 1μl of each is taken and mixed, and 5nl of the mixture is injected into the fertilized egg.

[0071] 6. Oviduct Transfer of Mouse Fertilized Eggs:

[0072] If the operation is less damaging, after one night of culture, generally more than 90% of the fertilized eggs will develop to the two-cell stage. These embryos can be transplanted into the fallopian tubes of pseudo-pregnant mice.

[0073] (1) Anesthetize the F0 surrogate mother (mated with E13.5 wild-type mice the night before):

[0074] (2) First, use an embryo transfer needle to transfer the two-cell stage embryos from M16 medium (sigma, Cat: M7292, 50 ml) to M2 medium (sigma, Cat: M7167, 50 ml), wash them once or twice, change to a new transfer tube, aspirate a small section of M2 medium, leave a bubble, aspirate another small section of M2 medium, leave a bubble, and then aspirate 10-15 injected two-cell embryos.

[0075] (3) Place the mother mouse on the operating table with her abdomen facing downwards. Wipe the back with alcohol. Make an incision of about 1 cm perpendicular to the longitudinal axis of the body in the middle of the head and tail of the mouse, near the back. A white fat pad will be visible. Pull it out with curved iris forceps to expose the ovary. Below the ovary is the fallopian tube. Under a microscope, use micro forceps to carefully tear open the capsule between the fallopian tube and the ovary. Use qualitative filter paper to absorb the surrounding blood. Find the opening (trumpet) of the fallopian tube and quickly insert the embryo transfer tube into it. When blowing in the embryo, avoid blowing in too much culture medium and paraffin oil, as shown in the figure below. Use the same method to perform embryo transplantation in the other fallopian tube.

[0076] (4) After the wound is sutured, the pseudopregnant mouse is kept warm and waited for it to wake up.

[0077] (5) Genotyping of mice was performed after birth.

[0078] The mice born were the F1 generation. The male and female individuals of the F1 generation were mated to obtain the F2 generation. The genome of the F2 generation was extracted and the amplified product was sent to the company for sequencing. The PCR amplified target gene fragment was obtained to obtain Cep44 + / - Heterozygous mice, Cep44 - / - Homozygous knockout mice.

[0079] Specifically:

[0080] A. Cut the mouse tail tissue and lyse it, and design 2 pairs of primers for PCR (the system is shown in Table 4).

[0081] F1: 5'-GCAGGATTCAGCAAGTCATTGTCTAC-3' (SEQ ID NO: 3);

[0082] R1: 5'-GGGTGCTTGTGTTAAGAAGGAGTAAC-3' (SEQ ID NO: 4);

[0083] F2: 5'-GGTCAATCGACGCAGAAGTTCTT-3' (SEQ ID NO: 5);

[0084] R2: 5'-CAGGACATCCTGGTATTGCAGTGA-3' (SEQ ID NO: 6);

[0085] The PCR reaction system for identifying genotypes is shown in Table 4 below:

[0086] Table 4

[0087]

[0088]

[0089] The PCR reaction program for identifying genotypes is shown in Table 5 below:

[0090] Table 5

[0091] temperature time Number of cycles 95℃ 3min 1 95℃ 15s 35 60℃ 15s 35 72℃ 30s 35 72℃ 5min 1 4℃ Always 1

[0092] B. Run the PCR products of the two primer pairs on 1% agarose gel. + / + No band appeared after amplification with primers F1 / R1 in wild mice, but a positive band appeared at 475 bp after amplification with primers F2 / R2. - / - After amplification with primers F1 / R1, a positive band appeared at 333 bp in homozygous knockout mice, but no band was found after amplification with primers F2 / R2. + / - Heterozygous mice showed positive bands at both locations. After amplification with primers F1 / R1, a positive band appeared at 333bp, and after amplification with primers F2 / R2, a positive band appeared at 475bp.

[0093] Among them, Cep44 - / - The CEP44 pathogenic gene knockout mouse model finally constructed in this application is a ciliopathy animal model.

[0094] The present application uses CRISPR / Cas9 technology to knock out mouse CEP44 exons 3-6 to obtain a mouse model with CEP44 pathogenic gene knockout. PCR results show that the target fragment of the mouse model with CEP44 pathogenic gene knockout is knocked out.

[0095] In order to further verify the knockout efficiency of the CEP44 pathogenic gene knockout mouse model, the embryonic proteins of wild-type mice and the CEP44 pathogenic gene knockout mouse model were extracted, and the protein levels of the knockout mice were detected by Western blotting experiments.

[0096] The results are as follows Figure 1 The results showed that wild-type mice had CEP44 protein, while the CEP44 pathogenic gene knockout mouse model lacked this band, confirming that the CEP44 pathogenic gene knockout mouse model was successfully prepared. Figure 1 As shown in A. Further analysis found that CEP44 knockout mice were embryonic lethal and exhibited ciliary disease-related phenotypes such as brain malformation, polydactyly and polycystic kidney.

[0097] This application uses PCR to identify Cep44 + / + Wild-type mice, Cep44 + / - Heterozygous mice and Cep44 - / - Genotyping of homozygous knockout mice (eg Figure 1 (shown in B).

[0098] Example 2: Phenotypic analysis of ciliopathy animal model

[0099] The ciliopathy animal model prepared in Example 1 was subjected to phenotypic analysis.

[0100] 1. Isolation and collection of mouse embryos:

[0101] A. Take Cep44 from 18.5 days of gestation + / - Pregnant mice were killed by cervical dislocation, and the embryos were removed and placed in ice-cold PBS;

[0102] B. Separate and remove the placentas and place them one by one in a 6-well plate filled with PBS;

[0103] C. According to the sequence numbering, a small amount of mouse tail tissue was cut and lysed, and its genotype was identified by PCR (the system is shown in Table 4);

[0104] D. Separate the tissues under a stereoscope for photographing or fixation.

[0105] 2. Use Alcian Blue and Alizarin Red S to stain fetal mouse bones:

[0106] A. Take Cep44 from 18.5 days of gestation + / - Pregnant mice were killed by cervical dislocation, and the embryos were removed and placed in PBS solution;

[0107] B. All skin on the embryonic body and limbs was removed, muscles around the chest and neck were removed, and internal organs were removed from the chest and abdominal cavities, followed by fixation with 95% ethanol for 14 days.

[0108] C. Embryos were stained with 0.01% Alzheimer's Blue 8GX for 24 hours.

[0109] D. Soak in 70% ethanol for 2 hours, 35% ethanol for 2 hours, 17.5% ethanol for 2 hours, and pure water until the sample is precipitated.

[0110] E. Soak in 1% potassium hydroxide for 24 hours.

[0111] F. Soak in 0.001% Alizarin Red S for 24 hours.

[0112] G. Soak in 1% potassium hydroxide 3 times, 2 hours each time.

[0113] H. The skeletons were cleared by immersion in 1% KOH / 20% glycerol for 24 h and finally stored in 50% ethanol / 50% glycerol.

[0114] I. Observation: Bone and cartilage can be stained red and blue respectively, and photographed using a Nikon digital camera and a stereo microscope.

[0115] 3. Paraffin sections of mouse tissues:

[0116] A. Sampling: Take out fresh tissue and fix it in 4% paraformaldehyde fixative for 24 hours, then use a scalpel to trim the tissue flat and store it in 70% alcohol;

[0117] B. Dehydration: Place the fixed samples in a white embedding box and place them in 70%, 80%, 95%, 100%, and 100% graded alcohol for 1 hour each;

[0118] C. Transparent: 50% alcohol and 50% xylene mixture, xylene for 0.5h each;

[0119] D. Wax dipping: immerse in 65℃ paraffin for 3 times, 1 hour each time;

[0120] E. Embedding: Pour the melted wax into the embedding frame, put the tissue in, label it, and trim the wax block;

[0121] F. Sectioning: After the wax block has cooled, clamp it on the paraffin sectioning machine stand, cut 4-6μm, place the slices on the water surface of the slide spreader (water temperature 40℃), pick up the tissue slices with a slide, bake them in a 60℃ oven for 1 hour, and use them for other experiments.

[0122] 4. Hematoxylin-eosin staining:

[0123] A. Baking: Bake in a 60℃ oven for 1 hour;

[0124] B. Dewaxing: Soak in xylene 3 times, 10 minutes each time;

[0125] C. Xylene removal: 100%, 100%, 100%, 95%, 80%, 75% gradient alcohol for 5 minutes each;

[0126] D. Washing: Wash with PBS 3 times, 5 minutes each time;

[0127] E. Hematoxylin staining: dilute the hematoxylin solution with deionized water at a ratio of 1:6, and then stain for 3-5 minutes. Use tap water to terminate the staining;

[0128] F. Differentiation: The stained sections were differentiated in 1% hydrochloric acid alcohol for 3-6 seconds;

[0129] G. Blueing: Place the differentiated slices in running water and allow them to blue for 10-30 minutes.

[0130] H. Eosin staining: Eosin staining solution was prepared with 95% alcohol in a ratio of 1:1 to make a working solution. Staining was performed for 1-5 minutes. After staining, the solution was washed with deionized water for 1-3 seconds.

[0131] I. Dehydration and transparency: 95%, 95%, 100%, 100% graded alcohol for 30 seconds each; xylene immersion 3 times, 3 minutes each time;

[0132] J. Sealing: Seal the slides with neutral resin, place in a fume hood to dry, and take photos.

[0133] Cep44 + / + Wild-type mice and Cep44 - / - Hematoxylin and eosin (H&E) staining of coronal sections of the heads of homozygous knockout mice at E14.5 (see Figure 2). Figure 1 Middle F).

[0134] Cep44 + / + Wild-type mice and Cep44 - / - Hematoxylin-eosin staining of coronal sections of embryonic kidneys of homozygous knockout mice at E18.5 (see Figure 2A). Figure 1 Middle G), the enlarged part of the dotted box is the glomerulus.

[0135] Cep44 + / + Wild-type mice and Cep44 - / - The cilia formation of renal tubular epithelial cells in homozygous knockout mice at E18.5 (e.g. Figure 1 Middle H), the dotted line indicates the renal tubule.

[0136] Cep44 + / + Wild-type mice and Cep44 - / -Kidney images of homozygous knockout mice at E18.5 (see Figure 2) Figure 1 Middle I), Cep44 - / - The mouse kidneys had an abnormal appearance.

[0137] 5. Isolation of primary MEFs:

[0138] A. Take Cep44 from 13.5 days of gestation + / - Pregnant mice were killed by cervical dislocation and immersed in 75% ethanol for 1 minute to ensure thorough disinfection;

[0139] B. Take out the embryos and place them in PBS, remove the placenta, separate the embryos, and place them one by one in a 6-well plate filled with PBS;

[0140] C. Number and cut off a small section of the tail for genotype identification;

[0141] D. Remove the embryo's head, internal organs and limbs, chop the remaining tissue repeatedly with a blade, add an appropriate amount of trypsin, and digest in a 37°C water bath for about 5-7 minutes.

[0142] E. Add an equal volume of DMEM medium to the cell turbid solution to terminate digestion, centrifuge and discard the supernatant;

[0143] F. Resuspend the dispersed cells in fresh complete medium and plant them into culture dishes for culture and passaging.

[0144] 6. Tissue protein extraction:

[0145] A. Using scissors, cut the tissue and snap freeze it in liquid nitrogen.

[0146] B. Add an appropriate amount of RIPA lysis buffer to the tissue and then grind it.

[0147] C. Centrifuge, collect the supernatant and discard the precipitate.

[0148] D. Determine the protein concentration using the BCA kit.

[0149] Tissue lysates were stained with antibodies against Cep44 and tubulin (Cep44 + / + Wild-type mice and Cep44 - / - Extracts from embryonic limbs of homozygous knockout mice were subjected to immunoblot analysis ( Figure 1 Middle C).

[0150] Cep44 + / + Wild-type mice and Cep44 - / - Whole embryo image of homozygous knockout mouse embryo at E18.5, Cep44 - / - Homozygous knockout mice are smaller, have extra fingers, abnormal head development, and systemic bleeding (such as Figure 1(middle D).

[0151] Cep44 + / + Wild-type mice and Cep44 - / - The number of fingers / toes of homozygous knockout mice at E18.5 (eg Figure 1 Middle E).

[0152] This application uses Alixin blue and alizarin red to test mice (control group and Cep44 - / - The results showed that the mouse model with CEP44 pathogenic gene knockout had abnormal skull, missing basesphenoid, occipital and anterior sphenoid bones, and cleft palate. At the same time, the present application also observed that CEP44 knockout mice had short limbs and multiple fingers / toes. Further analysis found that the sternum and vertebrae of CEP44 knockout mice were misaligned and the structure was abnormal. The results are as follows Figure 2 shown.

[0153] Cep44 + / + Wild-type mice and Cep44 - / - Whole embryo image of homozygous knockout mouse at E18.5 days, such as Figure 2 As shown in A. Cep44 + / + Wild-type mice and Cep44 - / - Staining of the craniofacial bones of homozygous knockout mice, e.g. Figure 2 As shown in B. + / + Compared with wild-type mice, Cep44 - / - Homozygous knockout mice show polydactyly, short limbs, and less mineralization of the phalanges (arrows). Figure 2 As shown in C. + / + Compared with wild-type mice, Cep44 - / - Homozygous knockout mice have vertebral segmentation (malalignment of vertebrae), such as Figure 2 As shown in D. Cep44 - / - The homozygous knockout mice have abnormal sternal fusion (sternum misalignment), such as Figure 2 As shown in E.

[0154] 7. Cell culture, transfection and synchronization:

[0155] 1) Cell culture:

[0156] The MEFs cells used in the present application were cultured in DMEM medium containing 10% fetal bovine serum, and the hTERT-RPE1 cells were cultured in F12 / DMEM medium containing 10% fetal bovine serum. The cells were placed in a 37° C. incubator with 5% CO 2 .

[0157] Lipofectamine 2000 or Lipofectamine 3000 was used to transfect hTERT-RPE1 cells with siRNA (siCEP44#1, siCEP44#2). The mixing ratio of siRNA and transfection reagent was determined according to the instruction manual (Invitrogen Lipofectamine TM 2000 transfection reagent, catalog number 11668030). After the transfection system is added to the cells (hTERT-RPE1 cells), the medium is replaced 5 hours later. The samples are collected 48-72 hours after cell transfection. MEFs cells do not need to be treated with siRNA.

[0158] siRNA was purchased from Qingke Biotechnology Co., Ltd.:

[0159] siCEP44#1:5'-GGCUAUAGUACAGCAUCAUTT-3';

[0160] siCEP44#2:5'-GGUGAUUCGUCACUUGUAUTT-3';

[0161] Control siRNA: 5'-UUCUCCGAACGUGUGUCACGU-3'.

[0162] 2) Immunofluorescence:

[0163] Fixation and perforation: Use cold methanol fixation and perforation method. Wash the coverslip with cells growing in the cell culture dish once with PBS, slowly add -20℃ methanol along the edge of the culture dish, and let it stand at -20℃ for 10 minutes to fix and perforate.

[0164] Pre-primary antibody wash: Remove cold methanol and wash coverslips 3 times with PBS for 10 minutes each, shaking slowly on a shaker.

[0165] Blocking before primary antibody: Remove the coverslip with forceps and invert the surface with cells in PBS containing 4% BSA for blocking at room temperature for 15-30 minutes.

[0166] Primary antibody incubation and washing: Then use the primary antibody diluted in PBS containing 4% BSA, and place the coverslip with cells upside down on the primary antibody, incubate at 4°C overnight or 37°C for 1 hour. Put the coverslip back into the cell culture dish with the cell side facing up, wash with PBS for a total of 3 times, 10 minutes each time, and place it on a shaker and shake slowly.

[0167] Blocking before secondary antibody: Remove the coverslip and place the side with cells upside down in PBS containing 4% BSA for blocking at room temperature for 15-30 minutes.

[0168] Secondary antibody incubation and washing: Then dilute the fluorescent secondary antibody with PBS containing 4% BSA and incubate at room temperature in the dark for 1 hour. Place the coverslip with the cell side facing up back into the cell culture dish, wash with PBS for a total of 3 times, 10 minutes each time, and place on a shaker and shake slowly.

[0169] Sealing: Drop DAPI solution prepared with 50% glycerol on a glass slide, place the coverslip with cells upside down on the DAPI solution, absorb the excess DAPI solution, seal the slide with nail polish, and observe with a fluorescence microscope after drying.

[0170] This application uses siRNA to knock down CEP44, and after labeling hTERT-RPE-1 cells with antibodies to the ciliary marker Arl13b (Cat No. 17711-1-AP, proteintech) and the centrosome marker γ-tubulin (Cat No. T6557, sigma), it was found that the proportion of Arl13b positive cells in the knockdown group hTERT-RPE-1 cells was significantly reduced compared with the control group, indicating that the proportion of cilia in hTERT-RPE-1 cells was significantly reduced after knocking down CEP44. Then, CEP44 knocked-out MEF cells were labeled, and the results showed that compared with wild-type cells, the proportion of cilia in CEP44 knocked-out MEF cells was significantly reduced. The results are as follows: Figure 3 shown.

[0171] Anti-Arl13b (green) and anti-γ-Tubulin (red) antibodies were used to perform immunofluorescence double labeling on the control group and Cep44 knockdown RPE-1 cells. Figure 3 As shown in A (DAPI (blue) labeled DNA).

[0172] The number of cilia in the control group and RPE-1 cells with Cep44 knockdown was counted and compared. Figure 3 As shown in B.

[0173] Anti-Arl13b (green) and anti-γ-Tubulin (red) antibodies were used to detect the control group, Cep44 - / - MEFs were double-labeled with immunofluorescence. Figure 3 As shown in C (DAPI (blue) labeled DNA).

[0174] The percentage of MEF cells growing cilia in the Cep44 control group and knockout group was counted and compared. Figure 3 As shown in D.

[0175] MEFs cells were cultured in serum-free medium for 24 h to induce primary cilia growth, and then 200 nM SAG (Cat. No. S7779, manufacturer selleck) was added to the serum-free medium for 20 h. The control group was treated with corresponding DMSO for 20 h, and the cells were collected.

[0176] Detection of Cep44 before and after SAG addition + / + and Cep44 - / - Ciliary localization of endogenous SMO in MEF cells. MEF cells were double-labeled with immunofluorescence using antibodies against Arl13b (green) and SMO (red), and DAPI (blue) was used to label DNA. Figure 3 As shown in E.

[0177] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. The use of gRNA of CEP44 pathogenic gene in preparing ciliopathy animal model, characterized in that: The accession number of the CEP44 pathogenic gene is: NM_001040157.3; The gRNA includes gRNA1 whose nucleotide sequence is shown in SEQ ID NO: 1 and gRNA2 whose nucleotide sequence is shown in SEQ ID NO:

2.

2. A method for constructing an animal model of ciliopathy, characterized in that: The ciliopathy animal model is obtained by knocking out the CEP44 pathogenic gene.

3. The method for constructing an animal model of ciliopathy according to claim 2, characterized in that: The ciliopathy animal model is obtained by knocking out exons 3 to 6 of the CEP44 pathogenic gene.

4. The method for constructing an animal model of ciliopathy according to claim 2 or 3, characterized in that: The following steps are involved: 1) in vitro transcribe gRNA1 with a nucleotide sequence as shown in SEQ ID NO: 1 and gRNA2 with a nucleotide sequence as shown in SEQ ID NO: 2 into mRNAs, respectively, to obtain active gRNA1 and gRNA2; 2) microinjecting the gRNA1, gRNA2, and Cas9 protein into mouse fertilized eggs to obtain F0 generation mice; 3) The F0 generation mice are mated with wild-type mice, and the F1 generation heterozygous mice are selected from the genotype identification results of the F1 generation mice. The heterozygous mice are mated to select homozygous mice to obtain a mouse model with CEP44 pathogenic gene knockout, that is, a ciliopathy animal model.

5. The method for constructing an animal model of ciliopathy according to claim 4, characterized in that: The step 3) comprises the following steps: According to exons 3-6 of the CEP44 pathogenic gene, PCR amplification was performed on F0 positive mice and wild-type mice using a primer set with sequences as shown in SEQ ID NOs: 3-6.

6. The method for constructing an animal model of ciliopathy according to claim 4, characterized in that: The concentrations of gRNA1 and gRNA2 are both 100 ng / μl.

7. Use of the method for constructing a ciliopathy animal model according to any one of claims 1 to 6 in screening for therapeutic drugs for ciliopathy.

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