CN118222568A - Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model - Google Patents
Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model Download PDFInfo
- Publication number
- CN118222568A CN118222568A CN202410210058.2A CN202410210058A CN118222568A CN 118222568 A CN118222568 A CN 118222568A CN 202410210058 A CN202410210058 A CN 202410210058A CN 118222568 A CN118222568 A CN 118222568A
- Authority
- CN
- China
- Prior art keywords
- wnt1
- animal model
- mutant gene
- osteogenesis imperfecta
- sgrna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000052547 Wnt-1 Human genes 0.000 title claims abstract description 57
- 101000781955 Homo sapiens Proto-oncogene Wnt-1 Proteins 0.000 title claims abstract description 55
- 238000010171 animal model Methods 0.000 title claims abstract description 46
- 206010031243 Osteogenesis imperfecta Diseases 0.000 title claims abstract description 36
- 238000003198 gene knock in Methods 0.000 title claims abstract description 23
- 238000010276 construction Methods 0.000 title abstract description 7
- 108091033409 CRISPR Proteins 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 230000035772 mutation Effects 0.000 claims abstract description 21
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 19
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 13
- 238000012216 screening Methods 0.000 claims abstract description 13
- 238000005516 engineering process Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 7
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 230000008685 targeting Effects 0.000 claims abstract description 4
- 235000013601 eggs Nutrition 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 238000011160 research Methods 0.000 claims description 10
- 238000009395 breeding Methods 0.000 claims description 7
- 230000001488 breeding effect Effects 0.000 claims description 7
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 230000008506 pathogenesis Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 241000282693 Cercopithecidae Species 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 238000010362 genome editing Methods 0.000 abstract description 5
- 230000003950 pathogenic mechanism Effects 0.000 abstract description 3
- 241000700159 Rattus Species 0.000 description 42
- 241000699670 Mus sp. Species 0.000 description 18
- 210000000988 bone and bone Anatomy 0.000 description 17
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 101150077398 WNT-1 gene Proteins 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000011892 Von Kossa's method Methods 0.000 description 4
- 238000003205 genotyping method Methods 0.000 description 4
- 230000013011 mating Effects 0.000 description 4
- 238000000520 microinjection Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 3
- 206010034156 Pathological fracture Diseases 0.000 description 3
- 208000005250 Spontaneous Fractures Diseases 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000002591 computed tomography Methods 0.000 description 3
- 210000003141 lower extremity Anatomy 0.000 description 3
- 210000003101 oviduct Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 206010017076 Fracture Diseases 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 108700020987 Wnt-1 Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033558 biomineral tissue development Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001054 cortical effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 101100156752 Caenorhabditis elegans cwn-1 gene Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 230000005971 DNA damage repair Effects 0.000 description 1
- 206010012559 Developmental delay Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 230000004156 Wnt signaling pathway Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000003275 diaphysis Anatomy 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002745 epiphysis Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 210000002082 fibula Anatomy 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108700029274 rat Wnt1 Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000012488 skeletal system development Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000001694 thigh bone Anatomy 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of biomedicine, discloses a construction method and application of a WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model, and particularly discloses a sgRNA of a specific targeting WNT1 mutant gene, wherein the sequence of the sgRNA is GCCGATGGTGGTAAGTGAGCTGG; or ACCAGCTCACTTACCACCATCGG; the mutation site of the WNT1 mutant gene is c104+1G > A. The sgRNA provided by the invention can be constructed based on CRISPR/Cas9 gene editing technology to obtain a knock-in animal model of WNT1 mutant gene (c.104+1G > A) for screening medicines for osteogenesis imperfecta diseases or researching pathogenic mechanisms of osteogenesis imperfecta diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a construction method and application of a WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model.
Background
The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nuclease) gene editing technology is a technology for targeted modification of a target gene by RNA-mediated Cas9 protein. Cas9 is CRISPR related nuclease, contains two nuclease domains, and can cut two DNA single strands of a target gene locus under the guidance of guide RNA, so that the DNA double strands are broken, and the autonomous DNA damage repair of cells is initiated. If exogenous DNA fragment carrying mutation is introduced into cells at the same time, the DNA fragment can be used as a template for repairing when the cells repair the autonomous DNA damage, thereby realizing multiple modifications such as fixed-point knocking-in of target sites, gene correction and the like.
The CRISPR/Cas9 technology has wide application in bioengineering and biomedicine, and can rapidly establish animal models and cell models of gene mutation for exploring the relationship between genetic variation and diseases.
Osteogenesis imperfecta (Osteogenesis Imperfecta, OI), a rare congenital skeletal development disorder characterized by fragile bones and frequent fractures, has a morbidity of about 1/20000, and can cause disability and death for severe cases. Mutations in the WNT1 gene may cause Osteogenesis Imperfecta (OI), however the pathogenesis is still unclear. The WNT1 protein encoded by the WNT1 gene belongs to a secreted glycoprotein and is one of the ligands of the WNT signaling pathway.
Disclosure of Invention
In clinical work, the inventor finds a child suffering from osteogenesis imperfecta, and the lower limb bones of the child are repeatedly fractured and deformed to be unable to stand, and the child is manifested as osteogenesis imperfecta. Further sequencing analysis is carried out on the genome DNA, and the WNT1 gene is found to be mutated, and c.104+1G > A. Based on the above, the inventor uses CRISPR/Cas9 technology to knock WNT1 mutant gene into rat osteogenesis imperfecta animal model according to mutation site c.104+1G > A of infant, which can provide effective experimental animal model for mechanism research of osteogenesis imperfecta, drug research and drug effect evaluation. Heretofore, there has been no report of constructing an animal model of bone insufficiency using rats and based on WNT1 mutant genes.
Aiming at the problems, the invention aims to provide a method for constructing a WNT1 mutant gene knock-in animal model based on a CRISPR/Cas9 technology and application thereof, and provides an effective experimental animal model for mechanism research of osteogenesis imperfecta, drug development and drug effect evaluation.
The object of the first aspect of the present invention is to provide an sgRNA specifically targeting WNT1 mutant gene.
The object of the second aspect of the invention is a kit.
The object of the third aspect of the present invention is to provide the use of the sgRNA of the first aspect of the present invention or the kit of the second aspect of the present invention for constructing a knock-in animal model of WNT1 mutant genes.
The fourth aspect of the invention aims to provide a method for constructing a WNT1 mutant gene knock-in animal model based on CRISPR/Cas9 technology.
The fifth aspect of the present invention is directed to a WNT1 mutant gene knock-in animal model.
The object of the sixth aspect of the present invention is to provide the use of the WNT1 mutant gene knockout animal model of the fifth aspect of the invention in screening drugs for preventing and/or treating osteogenesis imperfecta or research on the pathogenesis of osteogenesis imperfecta.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
In a first aspect of the present invention, there is provided an sgRNA specifically targeting WNT1 mutant gene, the sgRNA having a sequence of GCCGATGGTGGTAAGTGAGCTGG; or ACCAGCTCACTTACCACCATCGG; the mutation site of the WNT1 mutant gene is c104+1G > A.
In a second aspect of the invention there is provided a kit comprising the sgrnas of the first aspect of the invention.
In some embodiments of the invention, the kit further comprises Cas9 mRNA and a homology vector containing a mutation site that is c104+1g > a.
In some embodiments of the invention, the nucleotide sequence of the homologous vector containing the mutation site is shown in SEQ ID NO. 3.
In a third aspect, the invention provides the use of a sgRNA of the first aspect of the invention or a kit of the second aspect of the invention in the construction of a knock-in animal model for WNT1 mutant genes.
In some embodiments of the invention, the animal model is one of a mouse, a rat, a monkey.
In some preferred embodiments of the invention, the animal model is a rat.
In a fourth aspect of the present invention, there is provided a method for constructing a WNT1 mutant gene knock-in animal model based on CRISPR/Cas9 technology, the method comprising constructing by using the kit of the second aspect of the invention.
In some embodiments of the invention, the method comprises: introducing the mixture of the Cas9 mRNA, the homologous vector containing the mutation site and the sgRNA into fertilized eggs of model animals, obtaining surviving fertilized eggs, transplanting the surviving fertilized eggs into a surrogate mother body, breeding, obtaining offspring and carrying out genotyping screening.
In some embodiments of the invention, the concentration of Cas9 mRNA, the homologous vector containing the mutation site, and the sgRNA in the mixture is 200ng/μl, respectively.
In some embodiments of the invention, the means of introduction comprises injection.
In some embodiments of the invention, the genotyping screen comprises taking tail blood from a offspring animal, amplifying and sequencing by PCR.
In some preferred embodiments of the invention, the primers used in the PCR amplification comprise rCI-Rat Wnt1-F and rCI-Rat Wnt1-R primer pairs, the base sequence of the primer rCI-Rat Wnt1-F is shown as SEQ ID NO. 4, and the base sequence of the primer rCI-Rat Wnt1-R is shown as SEQ ID NO. 5.
In some preferred embodiments of the invention, the base sequence of the sequencing primer used in the sequencing process is shown in SEQ ID NO. 6.
In some embodiments of the invention, the surviving fertilized eggs are transplanted into a surrogate mother to be bred to obtain offspring, and the offspring is designated as an F0 generation animal model; mating the positive F0 generation animal model obtained through genotype identification and screening with a wild type, and breeding to obtain an F1 generation animal model; hybridizing the F1 generation heterozygote animal model obtained through genotyping and screening, breeding to obtain an F2 generation animal model, and genotyping and screening to obtain a positive F2 generation animal model, namely a WNT1 mutant gene knock-in animal model.
In some embodiments of the invention, the animal model is one of a mouse, a rat, a monkey.
In some preferred embodiments of the invention, the animal model is a rat.
In a fifth aspect of the present invention, there is provided a WNT1 mutant gene knock-in animal model constructed by the method of the fourth aspect of the invention.
In a sixth aspect, the present invention provides the use of the WNT1 mutant gene knock-in animal model of the fifth aspect of the invention in screening for a medicament for preventing and/or treating osteogenesis imperfecta or for studying the pathogenesis of osteogenesis imperfecta.
The beneficial effects of the invention are as follows:
The invention discovers that WNT1 gene is mutated in genome of children suffering from osteogenesis imperfecta for the first time, and the mutation site is c.104+1G > A. Therefore, based on the mutation site, the invention provides the sgRNA of the WNT1 mutant gene, and the sgRNA can be constructed based on a CRISPR/Cas9 gene editing technology to obtain the knock-in animal model of the WNT1 mutant gene (c.104+1G > A).
The invention constructs the WNT1 mutant gene (c.104+1G > A) knock-in animal model based on CRISPR/Cas9 gene editing technology, has the advantage of high knock-out efficiency, is beneficial to the pathogenic mechanism of osteogenesis imperfecta diseases and the application in the research and development of medicines for treating the osteogenesis imperfecta diseases, and can provide a new research direction for the related research of osteogenesis imperfecta. The invention constructs the WNT1 mutant gene knock-in rat for the first time, and has good medical clinical application prospect and great social benefit. And has wide industrial application value, and is a novel, advanced and practical new design.
The animal model constructed by the method has high controllability, good repeatability and easy breeding and feeding, overcomes the defects of the existing animal model, is more stable and more in line with clinical characteristics, and provides a good basis for pathogenesis and intervention research of osteogenesis imperfecta. By using the animal model, deeper insight can be provided for related researches of diseases related to osteogenesis imperfecta.
Drawings
FIG. 1 is a flow chart of the construction of a WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model using CRISPR/Cas 9.
FIG. 2 is an identification electrophoresis chart of PCR results of F0 generation WNT1 gene mutant knock-in rats.
FIG. 3 shows a WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model.
FIG. 4 shows that WNT1 mutant knock-in rats had a lower body weight than the control group.
FIG. 5 shows spontaneous fracture by X-ray film of hind limb of WNT1 mutant gene knock-in rat.
FIG. 6 shows CT scan and 3D reconstruction analysis of WNT1 mutant knock-in rat femur.
FIG. 7 shows the result of Vonkossa staining of the vertebrae of a rat animal model in which the WNT1 mutant gene was knocked in.
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.
Example 1
A method for establishing a WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model based on CRISPR/Cas9 gene editing technology comprises the following steps:
(1) Cas9/gRNA target design
The nucleotide sequence of the rat WNT1 gene is downloaded from NCBI database, and subjected to homology alignment, and the mutation site c.104+1G > A is determined on the homologous sequence of the rat according to the mutation site c.104+1G > A of the infant. Based on this site sequence, the sgRNA sequence was designed, including sgRNA1 and sgRNA2, wherein the nucleotide sequence of the sgRNA1 was 5'-GCCGATGGTGGTAAGTGAGCTGG-3' (SEQ ID NO: 1) and the nucleotide sequence of the sgRNA2 was 5'-ACCAGCTCACTTACCACCATCGG-3' (SEQ ID NO: 2).
(2) Construction of homologous recombination vector (Donor vector)
The homologous recombination vector Donor vector carries mutation sites, and the vector sequence is that 5'-TTGCTACTGGCACTGACCGCTCTGCCCGCAGCCCTGGGCGCCAACAGTAGTGGC CGATGGTGATAAGTGAGCTGGTACGGGGTCGCCACTTGTCTGGGGCAAAGAGCCA GGAACGGGCCCTA-3'(SEQ ID NO:3).
(3) In vitro transcription of sgrnas: the sgRNA is designed according to the target sequence, the construct ectotranscriptional vector pUC57-sgRNA is amplified and cultured to extract plasmid, and the plasmid is digested by DraI and recovered by glue. In vitro transcription was accomplished using the recovered product as template with T7 RNA polymerase and the product was gel recovered and purified, and finally the product was concentrated to 200 ng/. Mu.l by ethanol precipitation. And uniformly mixed with Cas9 mRNA and Donor vector according to the concentration ratio of 200 ng/. Mu.L to 200 ng/. Mu.L.
(4) Preparation of fertilized eggs: screening SD female mice of 3-4 weeks, and respectively injecting pregnant horse serum (PMSG) and chorionic gonadotrophin (hcg) at a time interval of 46-48 hours; mating female mice with adult male-fertile mice after HCG injection to fertilize the female mice; after euthanizing female mice the next day, fertilized eggs are collected from the oviduct and placed in a CO 2 incubator at 37 ℃ for later use.
(5) Prokaryotic microinjection, namely preparing microinjection injection needles and fixed needles; adding the prepared mRNA injection into a microinjection needle; screening fertilized eggs with normal morphology, placing the fertilized eggs in an injection dish, and injecting exogenous gene injection solution into fertilized egg nuclei by microinjection under an inverted microscope of 200-400 times; transferring the fertilized eggs after injection into an M16 culture medium, placing the fertilized eggs into a CO 2 incubator with constant temperature of 37 ℃ and 5%, culturing for 0.5-1 h, and transplanting.
(6) Preparation of surrogate mice and embryo transplantation
Pseudopregnant female mice were prepared: mating a fertile female mouse with a sterile male mouse after ligation of a seminiferous duct, and stimulating the female mouse to generate a series of gestation changes to obtain a pseudopregnant female mouse which is used as a female pregnant mouse after fertilized egg transgenosis; transplanting fertilized eggs injected with exogenous genes into oviducts of the surrogate female mice on the day of thrombus; placing the pregnant female mice in a clean cage box after transplantation, preserving heat, and placing the pregnant female mice back into the cage for feeding after the pregnant female mice are awake; after the oviduct transplantation is successful, the female mice generally farrowed 19-20 days after the operation; after the mice are born for 1 week, the number of the scissors can be carried out on the mice, and PCR identification can be carried out at the same time; mice were housed individually 3 weeks after birth.
(7) Identification of F0 mice at birth
Collecting 1-2 week old young mouse tissues (tail or toe tissues); lysing the tissue and extracting the genome;
PCR amplification and electrophoresis detection are carried out by specific primers aiming at target genes, and offspring of exogenous gene integration are screened out; the mice with integration are called naive mice (founder), can be passaged and established, and need to be identified for protein expression levels. The specific primer sequences for identifying the WNT1 mutant gene knock-in Rat genotype comprise rCI-Rat Wnt1-F (5'-CGCAGCGAGGTTAAGACCTGTTG-3', SEQ ID NO: 4) and rCI-Rat Wnt1-R (5'-CGCAGCGAGGTTAAGACCTGTTG-3', SEQ ID NO: 5), the sequencing primer is rCI-sequence-F (5'-GACAGCGAACCATGCTGCCT-3', SEQ ID NO: 6), the PCR amplification system is shown in Table 1, and the amplification program is shown in Table 2. Screening results are shown in figures 2-3, the amplified wild type sequence and the amplified mutant sequence are 594bp, the wild type sequence is shown in SEQ ID NO. 7, the WNT1 mutant sequence is shown in SEQ ID NO. 8, and the serial numbers of the F0 rats with positive passage #9, #10 and #27 are obtained through screening.
TABLE 1PCR amplification System
| Reaction components | Volume (mu L) |
| ddH2O | 21.85 |
| dNTP Mix | 1.5 |
| 10×PCR buffer | 3 |
| HotStart enzyme | 0.15 |
| Primer F | 1 |
| Primer R | 1 |
| DNA Template | 1.5 |
| Total | 30 |
TABLE 2PCR amplification procedure
SEQ ID NO:7(WT PCR product)：597bp
cgcaactataagaggcggtgcctcctgcagcggctgcttcagcccaccagccgggacagcgaaccatgctgcctgcggcccgcctccagactttctagagccagcctgagaatactcaccactgccctcaccgctgtgtccagtcccaccgtcgcggggagcaaccaaagtcgccagaaccgcagcacagaaccagcaaggccaggcaggccatggggctctgggcgctgctgcccagctgggtttctgctacgttgctactggcactgaccgctctgcccgcagccctgggcgccaacagtagtggccgatggtggtaagtgagctggtacggggtcgccacttgtcctggggcaaagagccaggaacgggccctacccagctcccacgctgtggggatcaccaacctacagactccctcctgtcctgtgacttcatatccagggtgctctcacctagaactagcccaaagacagaagctctgctggagtggggcaaatcacttgcatgcaaaaggccagctacaccaggctcagagaccatccccgtttaacacttccccgctatggcggagcaacaggtcttaacctcgctgcg
SEQ ID NO:8(Mutant PCR product)：597bp
cgcaactataagaggcggtgcctcctgcagcggctgcttcagcccaccagccgggacagcgaaccatgctgcctgcggcccgcctccagactttctagagccagcctgagaatactcaccactgccctcaccgctgtgtccagtcccaccgtcgcggggagcaaccaaagtcgccagaaccgcagcacagaaccagcaaggccaggcaggccatggggctctgggcgctgctgcccagctgggtttctgctacgttgctactggcactgaccgctctgcccgcagccctgggcgccaacagtagtggccgatggtgAtaagtgagctggtacggggtcgccacttgtcctggggcaaagagccaggaacgggccctacccagctcccacgctgtggggatcaccaacctacagactccctcctgtcctgtgacttcatatccagggtgctctcacctagaactagcccaaagacagaagctctgctggagtggggcaaatcacttgcatgcaaaaggccagctacaccaggctcagagaccatccccgtttaacacttccccgctatggcggagcaacaggtcttaacctcgctgcg, Wherein, the uppercase font is mutation site c.104+1G > A.
(8) And (3) mating the positive F0 generation rats which are up to 8 weeks old with wild type heterologous rats, breeding to obtain F1 generation rats, taking tail blood, and carrying out PCR amplification identification (the detailed process is the same as the step (3)) to obtain F1 generation heterozygote rats. Further hybridizing the F1 generation heterozygote rats which are up to 8 weeks old, breeding to obtain F2 generation rats, taking tail blood for PCR amplification identification (same as above), obtaining positive F2 generation homozygous rats, namely WNT1 mutant gene knock-in rats (WNT 1 mutant rats), and constructing to obtain a WNT1 mutant gene knock-in rats osteogenesis imperfecta animal model (figure 1).
Effect examples
1. Weighing body weight
Rats with 3 weeks old littermates WNT1 Wild Type (WT), heterozygotes (KI/+) and mutants (KI/KI, WNT1 mutant rats) constructed in example 1 were weighed.
The results showed that WNT1 mutant rats had significantly lower body weight than the wild type (fig. 4).
X-ray film detection
The fracture of the WNT1 mutant of 2 months old constructed in example 1 was examined by X-ray irradiation, and the bone length of the upper and lower limbs, width and circumference of epiphysis and diaphysis were measured by using the WNT1 wild type rat of the same nest as a control.
The results showed that the WNT1 mutant gene knocked-in rats had spontaneous fracture, and the phenotype of osteogenesis was observed in various sites such as tibia, fibula, femur, etc. (FIG. 5).
CT scan and 3D reconstruction analysis
Taking WNT1 wild type and WNT1 mutant rat thighbone in 2 month old same-nest F2 generation rats, performing mu CT scanning and 3D image reconstruction, observing a bone three-dimensional fine structure, and quantitatively analyzing parameters such as bone density, bone total amount, bone cortex thickness, bone cancellous thickness, number of bone trabeculae per millimeter, bone trabecula spacing and the like.
The results showed that the WNT1 mutant gene knocked in the cortical bone of the rat was thinned and bone in cancellous bone was reduced Liang Mingxian (fig. 6).
Vonkossa staining
The WNT1 mutant gene-knocked-in rats constructed in example 1 were euthanized, their vertebrae were removed, and hard tissue sections were stained with Vonkossa.
Vonkossa staining results showed that the WNT1 mutant gene constructed in example 1 significantly decreased mineralization ability of bone tissue in knock-in rats compared to wild type rats (fig. 7).
From the above results, it can be seen that the WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model constructed by the method of example 1 was able to successfully simulate human osteogenesis imperfecta disease. The homozygous mutation of the strain causes spontaneous fracture, the cortical bone becomes thin, and the trabecula in the cancellous bone is obviously reduced. The model has obviously reduced mineralization capacity of bone tissue when the pathogenesis research is carried out. In conclusion, the model can be used for researching the pathogenic mechanism of osteogenesis imperfecta caused by the WNT1 gene and has a possibly larger application value for the research and development of medicines for treating osteogenesis imperfecta.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. An sgRNA specifically targeting a WNT1 mutant gene, the sgRNA having a sequence of GCCGATGGTGGTAAGTGAGCTGG; or ACCAGCTCACTTACCACCATCGG; the mutation site of the WNT1 mutant gene is c104+1G > A.
2. A kit comprising the sgRNA of claim 1.
3. The kit of claim 2, further comprising Cas9 mRNA and a homology vector containing a mutation site, the mutation site being c.104+1g > a.
4. The kit according to claim 3, wherein the nucleotide sequence of the homologous vector containing the mutation site is shown in SEQ ID NO. 3.
5. Use of the sgRNA of claim 1 or the kit of any one of claims 2 to 4 for constructing a WNT1 mutant gene knock-in animal model.
6. The use according to claim 5, wherein the animal model is one of a mouse, a rat, a monkey.
7. A method of constructing a WNT1 mutant gene knock-in animal model based on CRISPR/Cas9 technology, comprising constructing by using the kit of claim 3 or 4.
8. The method according to claim 7, characterized in that the method comprises: introducing the mixture of the Cas9 mRNA, the homologous vector containing the mutation site and the sgRNA into fertilized eggs of model animals, obtaining surviving fertilized eggs, transplanting the surviving fertilized eggs into a surrogate mother body, breeding, obtaining offspring and carrying out genotype identification and screening.
9. A WNT1 mutant gene knock-in animal model constructed by the method of claim 7 or 8.
10. Use of the WNT1 mutant gene knockout animal model of claim 9 in screening for drugs for preventing and/or treating osteogenesis imperfecta or research on pathogenesis of osteogenesis imperfecta.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410210058.2A CN118222568A (en) | 2024-02-26 | 2024-02-26 | Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410210058.2A CN118222568A (en) | 2024-02-26 | 2024-02-26 | Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118222568A true CN118222568A (en) | 2024-06-21 |
Family
ID=91513133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410210058.2A Pending CN118222568A (en) | 2024-02-26 | 2024-02-26 | Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118222568A (en) |
-
2024
- 2024-02-26 CN CN202410210058.2A patent/CN118222568A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108660161B (en) | Method for preparing chimeric gene-free knockout animal based on CRISPR/Cas9 technology | |
| GB2578026A (en) | Method for constructing GLRX1 gene knock-out animal model based on CRISPR/CAS9 | |
| CN106191113A (en) | A kind of preparation method of MC3R gene knock-out pig | |
| CN105132427A (en) | Method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for method | |
| CN110951787A (en) | Immunodeficiency mouse, preparation method and application thereof | |
| CN108697067A (en) | Composition and method prepared by the organ of chimeric embryo-auxiliary | |
| CN106119284A (en) | A kind of product for building immunodeficient animals model and application thereof | |
| Kalds et al. | Redesigning small ruminant genomes with CRISPR toolkit: overview and perspectives | |
| JP2023116719A (en) | Disease model pig exhibiting stable phenotype, and production method thereof | |
| Park et al. | Assisted reproductive techniques and genetic manipulation in the common marmoset | |
| CN113088521A (en) | Construction method of Ahnak2 gene knockout animal model based on CRISPR/Cas9 technology | |
| CN111560401A (en) | Molecular breeding method for thickening interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala | |
| CN113234756A (en) | Construction method of LAMA3 gene knockout animal model based on CRISPR/Cas9 technology | |
| BG61041B1 (en) | Method for producing non human transgenetic animals by the introduction of exogenous dna in somatic embryonic animal cells | |
| CN114592075B (en) | Detection method of chimeric gonads after rice field eel germ cell xenograft and transplantation | |
| CN118222568A (en) | Construction method and application of WNT1 mutant gene knock-in rat osteogenesis imperfecta animal model | |
| CN111705063B (en) | ASGR1 mutant gene and application thereof in preparation of mammal liver injury sensitive model | |
| JP2009511010A (en) | Methods for transferring episomal vectors to animal cells | |
| CN109112162B (en) | Huntington's disease in-situ knock-in mouse model constructed by CRISPR/Cas9 technology and construction method | |
| CN107937439B (en) | Application of gene and construction method of animal model | |
| CN110129320A (en) | A kind of method obtaining gene editing sheep and its dedicated sgRNA and Oligo DNA | |
| CN113615638B (en) | Construction method and application of precocious sexual animal model | |
| WO2024143294A1 (en) | Genetically modified experimental animal production method by which mosaic modification is reduced or avoided | |
| Lian et al. | Application status of genome-editing tools in sheep and goats | |
| JPH03219821A (en) | Transgenic rat and production thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |