阅读说明：本技术 脊柱侧弯动物模型的建立方法 (Method for establishing scoliosis animal model ) 是由 舒玉琴 李璐 黎娟 郭子健 杨聪慧 许效玮 汤玉洁 贺佳欣 贺伟 于 2020-11-02 设计创作，主要内容包括：本申请实施例提供一种脊柱侧弯动物模型的建立方法,包括以下步骤：基于靶点基因,合成gRNA和Cas9 mRNA,靶点基因与ccdc57基因的外显子对应。将gRNA和Cas9 mRNA载入至斑马鱼的胚胎中,获得F0胚胎；F0胚胎培育至性成熟后与野生型斑马鱼交配获得F1胚胎,筛选有效突变胚胎培育至成年后获得相同基因型的有效敲除斑马鱼,将有效敲除斑马鱼进行自交获得F2胚胎,培育至成年筛选获得ccdc57纯合敲除斑马鱼。由于鱼类脊椎受到的流水阻力与人类直立行走脊椎所受重力相似,因此,斑马鱼建立的脊柱侧弯动物模型更适合对脊椎侧弯致病机理的挖掘和治疗方案的探究。(The embodiment of the application provides a method for establishing a scoliosis animal model, which comprises the following steps: gRNA and Cas9mRNA were synthesized based on a target gene corresponding to an exon of the cccd 57 gene. Loading the gRNA and Cas9mRNA into a zebrafish embryo to obtain an F0 embryo; cultivating the F0 embryo to sexual maturity, mating with wild zebra fish to obtain an F1 embryo, screening effective mutation embryos, cultivating to adults to obtain an effective knockout zebra fish with the same genotype, selfing the effective knockout zebra fish to obtain an F2 embryo, and cultivating to adults to obtain a cccd 57 homozygous knockout zebra fish. As the flowing water resistance of the spine of the fish is similar to the gravity of the spine of the human vertical walking, the scoliosis animal model established by the zebra fish is more suitable for exploring the pathogenesis of the scoliosis and researching the treatment scheme.)

1. A method for establishing a scoliosis animal model is characterized by comprising the following steps:

synthesizing gRNA and Cas9mRNA based on a target gene, wherein the target gene corresponds to an exon of a cccd 57 gene, and the sequence of the target gene is shown as SEQ ID NO. 1;

loading the gRNA and the Cas9mRNA into a zebrafish embryo, obtaining an F0 embryo;

cultivating an F0 embryo to sexual maturity, mating with wild zebra fish to obtain an F1 embryo, screening an effective mutation embryo, cultivating to adult to obtain an effective knockout zebra fish with the same genotype, selfing the effective knockout zebra fish to obtain an F2 embryo, and cultivating to adult to screen to obtain a cccd 57 homozygous knockout zebra fish.

2. The method of establishing an animal scoliosis model according to claim 1, wherein the synthesis of grnas comprises the steps of:

fusing a T7 promoter sequence, a target sequence and 20 base sequences before the gRNA scaffold to obtain a forward primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 2; designing 20 base sequences behind the gRNA scaffold as reverse primers, wherein the sequence of the reverse primers is shown as SEQ ID NO.3, and the sequence of the gRNA scaffold is shown as SEQ ID NO. 4;

taking pMD18-T plasmid inserted with gDNA fixed sequence as a template, and taking SEQ ID NO.2 and SEQ ID NO.3 as primers, carrying out first amplification to obtain gDNA;

gDNA was used as a template to synthesize gRNA.

3. The method for establishing a scoliosis animal model according to claim 2, wherein the first amplification step comprises: after a 2 minute hold at 94 ℃, 32 cycles were entered, each cycle undergoing 10 seconds of 98 ℃ denaturation, 30 seconds of 56 ℃ annealing, and 10 seconds of 68 ℃ extension.

4. The method of establishing an animal scoliosis model according to claim 1, wherein the Cas9mRNA synthesis comprises the steps of:

the plasmid pT3.Cas9-UTRGLOBIN is linearized by XbaI endonuclease, and the linearized plasmid is used as a template to synthesize Cas9 mRNA.

5. The method of establishing a scoliosis animal model according to claim 1, wherein the step of loading the gRNA and Cas9mRNA into zebrafish embryos comprises:

the gRNA and Cas9mRNA were mixed and diluted to final concentrations of 50ng/ul and 100ng/ul, respectively, and the mixture was injected into wild-type zebrafish embryos at 1-2 cell stage.

6. The method for establishing an animal model of scoliosis as claimed in claim 2, wherein said obtaining of F0 embryo and said breeding of F0 embryo to sexual maturity further comprises performing a knockout test on F0 embryo, said knockout test comprising:

and (3) culturing the F0 embryo to a membrane-out stage, picking the embryo, extracting genomes one by one for second amplification, and sequencing to detect the target sequence condition.

7. The method for establishing the scoliosis animal model according to claim 6, wherein in the second amplification, an F0 embryonic genome is used as a template, the sequence of a forward primer is shown as SEQ ID No.5, and the sequence of a reverse primer is shown as SEQ ID No. 6.

8. The method for creating an animal model of scoliosis according to claim 7, wherein said second amplification step comprises:

after 5 minutes at 94 ℃, 32 cycles were entered, each cycle undergoing denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, allowing the product to fully extend and complete the a-tailing process.

9. The knock-out detection method of claim 1, wherein the step of screening for an effective mutant embryo is:

culturing the F1 embryo to the membrane-out stage, picking out the embryo, extracting the genome one by one to carry out the third amplification, and determining the F1 embryo with non-triple mutation as the effective mutation embryo by sequencing.

10. The knock-out detection method of claim 1, wherein the step of breeding to adult screening for a homozygous knock-out zebrafish that can achieve cccd 57 is:

mating F1 zebra fish with the same non-triple mutation to obtain F2, culturing to adult, and then carrying out tail shearing screening to obtain homozygous zebra fish with non-triple base insertion/deletion, namely homozygous effective ccdc57 mutant zebra fish.

Technical Field

The application relates to the field of biomedicine, in particular to a method for establishing a scoliosis animal model.

Background

The scoliosis includes congenital scoliosis and idiopathic scoliosis, and Congenital Scoliosis (CS) is caused by abnormal development of vertebral bodies, and is often seen in vertebral body formation disorder and segmental defect, and the hemivertebral body deformity caused by the congenital scoliosis is the main cause of the congenital scoliosis. Idiopathic Scoliosis (IS) refers to scoliosis without a clear cause, which IS the most common cause of human curvature of the spine and affects about 3% of children worldwide, and the pathogenesis of the scoliosis IS not clear. Therefore, the treatment is limited to treatment of spinal deformities by performing extra-corporeal support or corrective surgery. Idiopathic scoliosis is common in adolescent and aggravates with age, has gender bias in the severity of the onset, and is often more rapid and severe in women with idiopathic scoliosis. Genomics association analyses have identified polymorphisms in different idiopathic scoliosis patient populations, and phenotypic and genetic heterogeneity makes it difficult to identify the specific genetic mutation that causes the pathology.

The scarcity of scoliosis animal models limits the study of idiopathic scoliosis pathology, the causative gene of scoliosis in humans, and the phenotype of scoliosis often not present in mouse knockout models. Rodents such as mice and the like and model animals such as rabbits, monkeys and the like are quadrupeds, and their spinal structures and kinematic biomechanics are different from those of humans. Humans have long been adapted to walk upright, and rodents forced to walk upright will severely stress the spine, resulting in spinal deformity. Therefore, a suitable animal for establishing the scoliosis animal model is urgently needed to be found.

Disclosure of Invention

The embodiment of the application aims to provide a method for establishing a scoliosis animal model so as to solve the technical problem of lack of the scoliosis animal model in the prior art.

In order to achieve the purpose, the technical scheme adopted by the application is as follows: the method for establishing the scoliosis animal model comprises the following steps:

synthesizing gRNA and Cas9mRNA based on a target gene, wherein the target gene corresponds to an exon of ccdc57 gene, and the sequence is shown as SEQ ID NO. 1;

loading the gRNA and Cas9mRNA into a zebrafish embryo to obtain an F0 embryo;

cultivating the F0 embryo to sexual maturity, mating with wild zebra fish to obtain an F1 embryo, screening effective mutation embryos, cultivating to adults to obtain an effective knockout zebra fish with the same genotype, selfing the effective knockout zebra fish to obtain an F2 embryo, and cultivating to adults to obtain a cccd 57 homozygous knockout zebra fish.

Preferably, synthesis of a gRNA comprises the steps of:

fusing a T7 promoter sequence, a target sequence and 20 base sequences in front of the gRNA scaffold to obtain a forward primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 2; designing 20 base sequences behind the gRNA scaffold as reverse primers, wherein the sequence of the reverse primers is shown as SEQ ID NO.3, and the sequence of the gRNA scaffold is shown as SEQ ID NO. 4;

taking pMD-18T plasmid inserted into gDNA scafffold as a template and SEQ ID NO.2 and SEQ ID NO.3 as primers, and carrying out first amplification to obtain gDNA;

gDNA was used as a template to synthesize gRNA.

Preferably, the first amplification step is: after a 2 minute hold at 94 ℃, 32 cycles were entered, each cycle undergoing 10 seconds of 98 ℃ denaturation, 30 seconds of 56 ℃ annealing, and 10 seconds of 68 ℃ extension.

Preferably, Cas9mRNA synthesis comprises the steps of:

the plasmid pT3.Cas9-UTRGLOBIN is linearized by XbaI endonuclease, and the linearized plasmid is used as a template to synthesize Cas9 mRNA.

Preferably, the step of loading the gRNA and Cas9mRNA into a zebrafish embryo comprises:

the gRNA and Cas9mRNA were mixed and diluted to final concentrations of 50ng/ul and 100ng/ul, respectively, and the mixture was injected into wild-type zebrafish embryos at 1-2 cell stage.

Preferably, obtaining the F0 embryo and breeding the F0 embryo to sexual maturity further comprises performing a knockout assay on the F0 embryo, the knockout assay comprising:

and (3) culturing the F0 embryo to a membrane-out stage, picking the embryo, extracting genomes one by one for second amplification, and sequencing to detect the target sequence condition.

Preferably, the F0 embryonic genome is used as a template in the second amplification, the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6.

Preferably, the step of second amplification is:

after 5 minutes at 94 ℃, 32 cycles were entered, each cycle undergoing denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, allowing the product to fully extend and complete the a-tailing process.

Preferably, the step of screening for an effective mutant embryo is:

culturing the F1 embryo to the membrane-out stage, picking out the embryo, extracting the genome one by one to carry out the third amplification, and determining the F1 embryo with non-triple mutation as the effective mutation embryo by sequencing.

Preferably, the step of breeding to an adult screening to obtain a ccdc57 homozygous knockout zebra fish is as follows:

mating the F1 zebra fish with the same non-triple mutation to obtain F2, and obtaining homozygous zebra fish with non-triple base insertion/deletion by tail-cutting screening, namely homozygous effective ccdc57 mutant zebra fish.

The method for establishing the scoliosis animal model provided by the embodiment of the application takes the ccdc57 gene as a target, and a target gene is designed on an exon of the ccdc57 gene. On the basis, chimera knockout F0 zebra fish, heterozygote knockout F1 zebra fish and homozygous F2 zebra fish are obtained. The homozygous F2 zebra fish shows the character of scoliosis and can be stably propagated and passaged. The method for establishing the scoliosis animal model is simple and reliable by establishing the scoliosis animal model by using the zebra fish. And because the flowing water resistance of the spine of the fish is similar to the gravity of the spine of the human vertical walking, the scoliosis animal model established by the zebra fish is more suitable for exploring the pathogenesis of the scoliosis and researching a treatment scheme.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.

Fig. 1 is a sequence diagram of ccdc57 knock-out target design and mutant strain targets: the ccdc57 knockout target is designed on an exon, and the specific sequence is shown as a red part in the figure. Line1(L1) and Line2(L2) are two knockout lines, wherein L1 Line target sequences lose 10 bases and L2 Line target sequences lose 4 bases;

fig. 2 is a side view and a back view of wild type and two ccdc57 knockout strains of zebra fish: the axis of the wild zebra fish is linear, and the axes of the two knockout strains of zebra fish are S-shaped or wavy;

FIG. 3 is a graph showing the skeletal characteristics of wild type and L1 zebra fish by Micro CT: E-F shows that the bones of the wild zebra fish are linear. G-L shows that bones of the zebra fish of the L1 knockout strain are in an s shape or a wave shape, wherein G-H bending degree is light, I-J bending degree is medium, and K-L bending degree is severe;

FIG. 4 is an appearance of wild type and L1 line embryos injected with ccdc57mRNA after back-filling: the wild-type zebrafish 72hpf embryo body axis is linear, and after normal ccdc57mRNA is injected, part of the embryo body axis is tilted to the back side. The L1 strain zebra fish 72hpf embryo body is bent towards the ventral side axially, and after the normal ccdc57mRNA is injected, the partial embryo body axis is straightened or tilted towards the dorsal side, which shows that the injection of the complemented normal ccdc57mRNA can effectively save the early stage axial ventral bending phenotype of the L1 strain embryo, and the scoliosis phenotype of the knocked-out strain is really caused by the deletion of the ccdc57 protein;

FIG. 5 is a graph of Q-PCR detection of wild type, L1 strain and anaplerotic L1 strain tail pressor peptides and their receptor expression levels: the expressions of L1 strain tail vasopressin 1 and 2 and the receptor thereof lacking ccdc57 protein are reduced, and the expressions of L1 strain tail vasopressin 1 and 2 and the receptor thereof can be obviously improved after excessive normal ccdc57mRNA is supplemented, which indicates that ccdc57 can regulate body axis development by influencing tail vasopressin signal path.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application clearer, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.

It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present application.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "a plurality" means two or more unless specifically limited otherwise.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, any reagents and materials used in the present invention are commercially available products or products which can be produced by a known method.

When a proper animal is found to establish the scoliosis animal model, the animal used for research should have the same or similar motion biomechanics as human beings, the flowing water resistance of the fish spine is similar to the gravity of the human upright walking spine, and inheritable scoliosis is easy to occur in the fish culture process, so the fish is more suitable for establishing the scoliosis disease model than quadrupeds.

In 2016, there was a first paper that suggested that spinal fluid flow impairment due to defects in cilia development could cause scoliosis in zebrafish. In 2019, it was reported that cerebrospinal fluid flow disturbance further analyzed that the adrenergic signals transmitted to nerve cells in the spinal canal were reduced, thereby affecting the contraction stimulation of the slow muscles in the back by the downstream urocortin signal pathway, resulting in reduced spinal dorsal contractility and body axial ventral curvature due to unbalanced dorsal and ventral stress.

CCDC57 belongs to the structural family of Coiled-coil domains (CCDC), and reports related to functions of CCDC are not found before 2019. Partial gene associations in the CCDC family have been reported to be associated with ciliary developmental regulation, with CCDC114, CCDC103, CCDC65, CCDC39/40 mutations being shown to result in primary ciliary dyskinesia. Gheirtanmd first suggested in 2019 that CCDC57 was one of the centrosome-associated interaction components, and may be associated with centrosome replication. On the one hand, the CCDC57 protein can recruit CEP63, CEP152 and PLK4 molecules to gather to a centrosome. CCDC57 protein, on the other hand, localizes on central body microtubules and interacts with tubulin, affecting microtubule assembly, stabilization and mitotic progression. In general, CCDC57 protein is a pleiotropic factor that regulates centromere replication, and is able to further regulate cilia formation and mitotic progression. The inventor finds that CCDC57 is expressed in the ventricles of the zebra fish in the research process, and the ventricles are communicated with spinal nerves and are fully provided with cilia, which suggests that the zebra fish CCDC57 may be involved in the regulation of the development of the cilia in the ventricles.

On the basis, the method for establishing the scoliosis animal model is designed, and comprises the following steps:

synthesizing gRNA and Cas9mRNA based on a target gene, wherein the target gene corresponds to an exon of ccdc57 gene, and the sequence is shown as SEQ ID NO. 1;

loading the gRNA and Cas9mRNA into a zebrafish embryo to obtain an F0 embryo;

cultivating the F0 embryo to sexual maturity, mating with wild zebra fish to obtain an F1 embryo, screening effective mutation embryos, cultivating to adults to obtain an effective knockout zebra fish with the same genotype, selfing the effective knockout zebra fish to obtain an F2 embryo, and cultivating to adults to obtain a cccd 57 homozygous knockout zebra fish.

The method for establishing the scoliosis animal model mainly comprises two parts of constructing screening of a ccdc57 gene knockout zebra fish and a ccdc57 effective mutation strain.

The ccdc57 gene knockout zebra fish can be constructed in the following way: obtaining a zebra fish ccdc57 Gene sequence (Gene ID:100318317) from NCBI database (https:// www.ncbi.nlm.nih.gov/Gene/100318317); aiming at the first exon region design target of the ccdc57 gene, according to the target design rule, selecting a 16-24bp sequence with the beginning and the end of GG/GGG being close to NGG as the target gene, wherein the sequence is specifically shown as SEQ ID NO.1 and comprises the following steps: GGAGGAAAGGGACAAAGAGC are provided.

When the target gene was determined, gRNA and Cas9mRNA were synthesized and loaded into zebrafish embryos, as by microinjection into embryos, to obtain F0 embryos. F0 zebra fish is a knockout chimera of the ccdc57 gene.

F0 embryo is cultured until sexual maturity, and the zebra fish is mated with wild zebra fish to obtain F1 embryo. F1 embryos include both efficiently mutated embryos and inefficiently mutated embryos, and therefore it is desirable to screen efficiently mutated embryos from them and breed them into adults. A validly mutated F1 zebrafish is a knockout heterozygote of the ccdc57 gene, i.e., one of the two copies of the gene is mutated by frame shift.

The effective mutated embryo in the F1 embryo may have different genotypes, the effective knockout zebra fish of the same genotype is selfed to obtain an F2 embryo, and the F2 zebra fish is a cccd 57 homozygous knockout zebra fish, namely the zebra fish with two copies of the gene having frame shift mutation.

According to the method for establishing the scoliosis animal model, the inventor finds that ccdc57 is expressed in the ventricles of the zebra fish, and cilia are distributed in the ventricles communicated with spinal nerves, so that the fact that the ccdc57 of the zebra fish possibly relates to the regulation and control of the development of the cilia in the ventricles is suggested. Targeting ccdc57 gene, designing target gene on its exon. On the basis, chimera knockout F0 zebra fish, heterozygote knockout F1 zebra fish and homozygous F2 zebra fish are obtained. The homozygous F2 zebra fish shows the character of scoliosis and can be stably propagated and passaged. The method for establishing the scoliosis animal model is simple and reliable by establishing the scoliosis animal model by using the zebra fish. And because the flowing water resistance of the fish spine is similar to the gravity of the human vertical walking spine, the scoliosis animal model established by the zebra fish is more suitable for the research of idiopathic scoliosis pathology.

In one embodiment, synthesis of a gRNA comprises the steps of:

fusing a T7 promoter sequence, a target sequence and 20 base sequences in front of the gRNA scaffold to obtain a forward primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 2; designing 20 base sequences behind the gRNA scaffold as reverse primers, wherein the sequence of the reverse primers is shown as SEQ ID NO.3, and the sequence of the gRNA scaffold is shown as SEQ ID NO. 4;

taking pMD18-T plasmid inserted with gDNA fixed sequence as a template, and taking SEQ ID NO.2 and SEQ ID NO.3 as primers, carrying out first amplification to obtain gDNA;

gDNA was used as a template to synthesize gRNA.

The synthesis of a particular gRNA can be accomplished by designing and synthesizing primers for expanding the gDNA template after the target gene has been identified. By directly synthesizing a long fragment primer, fusing a T7 promoter sequence and a target sequence required by transcription with 20 base sequences before the gRNA scaffold to design a forward primer; the 20 base sequences after the gRNA scaffold were designed as reverse primers.

A forward primer: TAATACGACTCACTATAGGGAGGAAAGGGACAAAGAGCGTTTTAGAGCTAGAAATAGC (SEQ ID NO. 2);

reverse primer: AGCACCGACTCGGTGCCACT (SEQ ID NO. 3).

Then, the pMD18-T plasmid inserted with gRNA scaffold is taken as a template, and SEQ ID NO.2 and SEQ ID NO.3 are taken as primers to carry out the first amplification, for example, the amplification is carried out by adopting a PCR mode.

After PCR amplification is finished, a T7 promoter connecting target and a gDNA template fragment of gRNA scaffold can be obtained, a target band can be separated through electrophoresis, and further purification and recovery are carried out through a gel recovery kit (Omega D2500-01) to obtain the gDNA template fragment. After the gDNA template fragment is obtained, a gRNA can be synthesized by using a T7 high-efficiency transcription kit, and then the gRNA is purified by using a LiCl precipitation method.

In one embodiment, the first amplification step is: after a 2 minute hold at 94 ℃, 32 cycles were entered, each cycle undergoing 10 seconds of 98 ℃ denaturation, 30 seconds of 56 ℃ annealing, and 10 seconds of 68 ℃ extension.

The PCR comprises the following specific steps: a50 ul reaction system was prepared by amplifying a pMD18T-gRNA plasmid as a template, SEQ ID NO.2 and SEQ ID NO.3 as primers and KOD-FX Hi-Fi (Phanta Super-Fidelity DNA Polymerase), and included 25ul 2 XPCR buffer, 10ul 2mM dNTPs, 1.5ul forward primer, 1.5ul reverse primer, 2ul 400ng/ul template plasmid, and 1ul KOD-FX Hi-Fi. The three-step method is adopted as follows: after 2 minutes at 94 °, 32 cycles were entered, each cycle undergoing 10 seconds of 98 ° denaturation, 30 seconds of 56 ° annealing, and 10 seconds of 68 ° extension.

In one embodiment, Cas9mRNA synthesis comprises the steps of:

the plasmid pT3.Cas9-UTRGLOBIN is linearized by XbaI endonuclease, and the linearized plasmid is used as a template to synthesize Cas9 mRNA.

The Cas9 linearized template can be purified first, and then the Cas9mRNA can be synthesized by taking the purified linearized plasmid as a template. The linearized product from Cas9 can be purified by phenol chloroform extraction.

The Cas9 template was linearized, and Cas9mRNA and Cas9mRNA were synthesized using a T3 mRNA synthesis Kit (mmesse mmachinet mt3 Transcription Kit, AM 1348).

After Cas9mRNA is synthesized under the above conditions, Cas9mRNA can also be purified by LiCl precipitation

In one embodiment, the step of loading gRNA and Cas9mRNA into zebrafish embryos comprises:

the gRNA and Cas9mRNA were mixed and diluted to final concentrations of 50ng/ul and 100ng/ul, respectively, and the mixture was injected into wild-type zebrafish embryos at 1-2 cell stage.

Wherein, the purified gRNA and Cas9mRNA are mixed and diluted according to a certain proportion, so that the final concentration is 50ng/ul and 100ng/ul respectively, such as ccdc57_ gRNA (100ng/ul, 3ul) and Cas9mRNA (400ng/ul, 3ul) are mixed and diluted according to the ratio of 1: 1 to obtain ccdc57_ gRNA and Cas9mRNA mixtures with final concentrations of 50ng/ul and 100ng/ul, respectively. And injecting the RNA mixture into the zebra fish embryo at the 1-2 cell stage by using a P830 microinjection instrument of WPI company to obtain a knockout F0 embryo.

In one embodiment, obtaining between the F0 embryo and the F0 embryo culture to sexual maturity further comprises performing a knockout test on the F0 embryo, the knockout test comprising:

and (3) culturing the F0 embryo to a membrane-out stage, picking the embryo, extracting genomes one by one for second amplification, and sequencing to detect the target sequence condition.

Before the F0 embryo is cultured to reach sexual maturity, the knockout detection of the F0 embryo is necessary to detect the effective knockout condition of the F0 embryo. If the knockout F0 embryo is cultured to the membrane-out stage, a certain number of embryos are randomly picked, the genomes are extracted one by one to carry out the second amplification such as PCR amplification, and the target sequence condition is detected by sequencing. For example, the NaOH method can be used to rapidly extract the genome.

In one example, the F0 embryonic genome was used as the template in the second amplification, the sequence of the forward primer is shown in SEQ ID NO.5, and the sequence of the reverse primer is shown in SEQ ID NO. 6.

The target sequence detection can utilize NCBI online blast software to design a primer for detecting a target fragment, the target distance is kept above 120bp from upstream and downstream primers, and a PCR product is controlled within 400 and 1000 bp. The detection primer sequences are as follows:

a forward primer: TGCTGGAAGCTGTGGACCTG (SEQ ID NO.5)

Reverse primer: CTGCTCCTCTAGGGCATTCTG (SEQ ID NO.6)

In one embodiment, the second amplification step is:

after 5 minutes at 94 ℃, 32 cycles were entered, each cycle undergoing denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, allowing the product to fully extend and complete the a-tailing process.

The second amplification can be performed by PCR, wherein each 20ul of the PCR system comprises 10ul of PCR Mix, 1ul of forward primer (10uM) shown as SEQ ID NO.5, 1ul of reverse primer (10uM) shown as SEQ ID NO.6, and 2ul of genome template (extracted by the above NaOH method). The general PCR program is adopted for amplification, and the specific program is as follows: after 5 minutes at 94 ℃, 32 cycles were entered, each cycle undergoing denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, allowing the product to fully extend and complete the a-tailing process. The whole amplification process lasts for about 1 hour and 30 minutes, 2ul of samples are taken for electrophoresis after the amplification is finished, and the remaining samples are sequenced after the target fragments are determined to be amplified.

In one embodiment, the step of screening for a valid mutant embryo is:

culturing the F1 embryo to the membrane-out stage, picking out the embryo, extracting the genome one by one to carry out the third amplification, and determining the F1 embryo with non-triple mutation as the effective mutation embryo by sequencing.

After cultivation for about 3 months, F0 zebra fish can be mated with wild zebra fish to obtain F1 embryo. After hatching out the membrane, randomly selecting a certain number of embryos, quickly extracting the genome by adopting an NaOH method, namely correspondingly adding 50ul of NaOH solution into a single embryo with the concentration of 50mM, digesting at 95 ℃ for 10-15 minutes, uniformly shaking, uniformly mixing, heating for 5 minutes, taking out, standing, cooling, adding 5ul of Tris-HCl solution (with the concentration of 1M and the pH value of 8.0), and uniformly mixing to obtain the PCR template. Determining whether F1 embryos have non-triple after sequencing, breeding F1 embryo groups containing non-triple mutation to adults, and screening non-triple mutation F1 individuals by tail cutting, namely: cutting the tails of adult F1 groups one by one, adding 100ul NaOH solution into one sample correspondingly, heating and digesting uniformly, adding 10ul Tris-HCl solution, and mixing uniformly to be used as a PCR template. After the detection fragment is amplified, sequencing is carried out to confirm the genotype.

In one embodiment, the step of breeding to an adult screening to obtain a ccdc57 homozygous knockout zebra fish is as follows:

mating the F1 zebra fish with the same non-triple mutation to obtain F2, and obtaining homozygous zebra fish with non-triple base insertion/deletion by tail-cutting screening, namely homozygous effective ccdc57 mutant zebra fish.

F1 produced by the same piece of F0 may contain multiple mutations, and needs to be screened and confirmed, and the same mutation is classified into a line to be selfed to obtain F2. Meanwhile, F2 is composed of 1/4+ +, 1/4 and 1/2+, wherein 1/4 is a homozygous and effective mutant, so that F2 needs to be further screened for homozygous zebrafish.

Example 1

1) Construction of ccdc57 Gene knockout zebra fish

1-1) obtaining a zebra fish ccdc57 Gene sequence (Gene ID:100318317) from NCBI database (https:// www.ncbi.nlm.nih.gov/Gene/100318317); aiming at the first exon region design target of the ccdc57 gene, according to the target design rule, selecting a 16-24bp sequence with the beginning and the end of GG/GGG being close to NGG as the target, wherein the sequence is specifically shown as SEQ ID NO.1 and comprises the following steps: GGAGGAAAGGGACAAAGAGC, respectively;

1-2) design and synthesis of primers for expanding gDNA templates. By directly synthesizing a long fragment primer, fusing a T7 promoter sequence and a target sequence required by transcription with 20 base sequences before the gRNA scaffold to design a forward primer; the 20 base sequences after the gRNA scaffold were designed as reverse primers. Specifically shown as SEQ ID NO.2 and SEQ ID NO.3, the sequence is as follows:

a forward primer: TAATACGACTCACTATAGGGAGGAAAGGGACAAAGAGCGTTTTAGAGCTAGAAATAGC (SEQ ID NO. 2);

reverse primer: AGCACCGACTCGGTGCCACT (SEQ ID NO. 3).

1-3) amplification and purification of gDNA templates: a50 ul reaction system was prepared by amplifying a pMD18T-gRNA plasmid as a template, SEQ ID NO.2 and SEQ ID NO.3 as primers and KOD-FX Hi-Fi (Phanta Super-Fidelity DNA Polymerase), and included 25ul 2 XPCR buffer, 10ul 2mM dNTPs, 1.5ul forward primer, 1.5ul reverse primer, 2ul 400ng/ul template plasmid, and 1ul KOD-FX Hi-Fi. The three-step method is adopted as follows: after 2 minutes at 94 °, 32 cycles were entered, each cycle undergoing 10 seconds of 98 ° denaturation, 30 seconds of 56 ° annealing, and 10 seconds of 68 ° extension.

After PCR amplification is completed, a T7 promoter connection target and a gDNA template fragment of gRNA scaffold can be obtained, a target band is separated through electrophoresis, and further purification and recovery are carried out through a gel recovery kit (Omega D2500-01), and the specific steps are as follows: 1) the agarose gel containing the target fragment was cut under an ultraviolet lamp and transferred to an EP tube, and 400. mu.l Binding Buffer was added; 2) placing the EP tube in a 65 ℃ water bath kettle, and heating until the gel block is completely melted; 3) assembling the DNA recovery and purification column and a 2ml collecting tube, adding 300 mul Binding Buffer, centrifuging at 13000g for 1 minute, and removing the filtrate; 4) adding the solution in the step 2) into a recovery column, centrifuging for 1 minute at the room temperature of 13000g, and removing the filtrate; 5) 700ul of the eluate was added to the recovery column, and the mixture was centrifuged at 13000g for 1 minute at room temperature, and the filtrate was discarded. The step can be repeated for 1-2 times; 6) centrifuge at 15000g for 2 minutes at room temperature, discard filtrate and collection tube, open recovery column lid, air-dry for 2 minutes. 7) The purification column was mounted on a RNase-free 1.5ml EP tube, 30ul DEPC water was added, and the mixture was allowed to stand for 2 to 3 minutes and centrifuged at 13000g for 1 minute at room temperature. The recovery efficiency can be improved by repeating the step once; 8) and (4) detecting whether the product is degraded or not by glue running, and storing the glue recovery product in a refrigerator at the temperature of-20 ℃ after the concentration is determined.

1-4) gRNA synthesis and purification: gDNA was synthesized using a T7 High-efficiency Transcription Kit using gDNA as a template, and the synthesis procedure was described with reference to a T7 RNA High-efficiency synthesis Kit (TranscriptAId T7 High Yield Transcription Kit, K0441): the 20ul synthesis system included 4ul 5 × Transcript Aid Reaction Buffer, 8ul 2.5mM NTP Mix, 2ul Transcript Aid Enzyme Mix, 1ug cccdc 57_ gDNA. After reacting for 2-4 hours at 37 ℃, adding DNase for treating for 15 minutes, adding 30ul DEPC water and 30ul 7.5M LiCl solution, mixing uniformly, and standing at-20 ℃ for precipitation overnight. The next day, centrifugation was carried out at 13200rmp 4 ℃ for 10 minutes, the supernatant was removed, 700ul of 75% ethanol solution was added for washing, centrifugation was carried out at 13200rmp 4 ℃ for 5 minutes, the supernatant was removed, the lid was opened until complete drying, and 50ul of DEPC water was added for dissolution. After measuring the concentration by a spectrophotometer, ccdc57_ gRNA was diluted to 100ng/ul, 3ul per tube was aliquoted and stored at-80 ℃.

1-5) Cas9mRNA synthesis and purification:

cas9 linearized template preparation: the pT3. Cas9-UTRGOBin plasmid was linearized with Fast-XbaI endonuclease, and 50ul of the digestion system consisting of 5ul of 10xbuffer, 1ul of XbaI endonuclease and 5ug of plasmid was digested in a water bath at 37 ℃ for 2 hours.

Cas9 linearized template purification: the obtained linearized product was purified by phenol chloroform extraction: adding 50 mul of enzyme digestion system with water to 100 mul, adding isovolumetric equilibrium phenol/chloroform, mixing uniformly, centrifuging for 5 minutes at 7500g, taking the upper aqueous phase, adding one tenth volume of NaAC and 2 times volume of absolute ethyl alcohol, settling for 2 hours at-20 ℃, centrifuging at 4 ℃, centrifuging for 15 minutes at 13000g, removing the supernatant, adding 700ul 75% ethanol, centrifuging for 5 minutes at 13000g, removing the supernatant, keeping the sediment, opening an ep tube cover, drying in the air, adding DEPC water for dissolving, measuring the concentration by a spectrophotometer, and storing in a refrigerator at-20 ℃.

Cas9mRNA synthesis and purification:

the linearized Cas9 template was obtained in the above steps, and Cas9mRNA was synthesized using T3 mRNA synthesis Kit (mmesse mmachienetmt 3 Transcription Kit, AM1348), the synthesis steps being described with reference to the Kit: the 20ul synthetic system included 4ul 5 × Transcript Aid Reaction Buffer, 10ul 2 × NTP Mix, 1ul Transcript Aid Enzyme Mix, 1ug Cas9 linearized plasmids. The reaction is carried out for 2 hours at 37 ℃, and whether mRNA is synthesized or degraded is detected by electrophoresis.

Cas9mRNA purification: the reaction system was treated with DNase for 15 min to remove the plasmid template, 30ul DEPC water and 30ul 7.5M LiCl solution were added, mixed well and precipitated at-20 ℃ overnight. The next day, centrifugation was carried out at 13200rmp 4 ℃ for 10 minutes, the supernatant was removed, 700ul of 75% ethanol solution was added for washing, centrifugation was carried out at 13200rmp 4 ℃ for 5 minutes, the supernatant was removed, the lid was opened until complete drying, and 50ul of DEPC water was added for dissolution. After measuring the concentration by spectrophotometer, Cas9mRNA was diluted to 200ng/ul, 3 ul/tube split and stored in-80 ℃ refrigerator.

1-6) microinjection to obtain knockout F0 embryos: ccdc57_ gRNA (100ng/ul, 3ul) and Cas9mRNA (400ng/ul, 3ul) were expressed as 1: 1, obtaining ccdc57_ gRNA and Cas9mRNA mixtures with final concentrations of 50ng/ul and 100ng/ul respectively, and injecting the RNA mixtures into zebra fish embryos at a 1-2 cell stage by utilizing a P830 microinjection instrument of WPI company to obtain knockout F0 embryos.

1-7) F0 embryo knockout efficiency estimation:

and (3) extracting a genome: culturing the knockout F0 embryos to a membrane-out stage, randomly picking 15 embryos, dividing into 3 groups, rapidly extracting genome by using NaOH method, namely adding 100ul NaOH solution into each group of embryos correspondingly, wherein the concentration is 50mM, digesting at 95 ℃ for 10-15 minutes, shaking, uniformly mixing, heating for 5 minutes, taking out, standing, cooling, adding 10ul Tris-HCl solution (the concentration is 1M, and the pH is 8.0), and uniformly mixing to obtain the PCR template.

And (3) target point sequence detection: the primers for detecting the target fragments are designed by using NCBI online blast software, the target distance is kept above 120bp from upstream and downstream primers, and the PCR product is controlled within 400-1000 bp. The detection primer sequences are as follows:

a forward primer: TGCTGGAAGCTGTGGACCTG (SEQ ID NO.5)

Reverse primer: CTGCTCCTCTAGGGCATTCTG (SEQ ID NO.6)

The PCR system included 10ul PCR Mix, 1ul forward primer (10uM), 1ul reverse primer (10uM), and 2ul genomic template (extracted by NaOH method as described above) per 20 ul. The general PCR program is adopted for amplification, and the specific program is as follows: after 5 minutes at 94 ℃, 32 cycles were entered, each cycle undergoing denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, allowing the product to fully extend and complete the a-tailing process. The whole amplification process lasts for about 1 hour and 30 minutes, 2ul of samples are taken for electrophoresis after the amplification is finished, and the remaining samples are sequenced after the target fragments are determined to be amplified.

2) Screening of ccdc57 effective mutant strains

2-1) obtaining F1 heterozygous effective mutants: after cultivation for about 3 months, F0 zebra fish can be mated with wild zebra fish to obtain F1 embryo.

The F1 embryo adopts a mode of 1-7) F0 embryo knockout efficiency estimation steps to extract genome and target sequence detection, and a detection fragment is amplified by PCR and sequenced. Determining whether F1 embryos have non-triple after sequencing, breeding F1 embryo groups containing non-triple mutation to adults, and screening non-triple mutation F1 individuals by tail cutting, namely: cutting the tails of adult F1 groups one by one, extracting genome and target point sequence detection of one sample by adopting a mode of 1-7) F0 embryo knockout efficiency estimation steps, and sequencing after detecting fragments are obtained through PCR to confirm the genotype. Two lines, L1 and L2, were selected that exhibited different sequences in the ccdc57 designed target region. As shown in fig. 1, ccdc57 knock-out target design and mutant strain target sequences: the ccdc57 knockout target is designed on the exon, and the specific sequence is as the sequence marked by the grey part of WT (Wild Type, namely Wild), as shown in SEQ ID NO. 20. Line1(L1) and Line2(L2) are two knockout lines in which the L1 Line target sequence is missing 10 bases (lighter gray off-white portion) and its sequence is shown in SEQ ID No. 21. The L2 strain target sequence is missing 4 bases (gray part), and the sequence is shown in SEQ ID NO. 22.

2-2) homozygous efficient mutants of F2: mating the F1 zebra fish with the same non-triple base insertion/deletion to obtain F2, and carrying out tail shearing screening to obtain homozygous zebra fish with non-triple base insertion/deletion, namely homozygous effective ccdc57 mutant zebra fish. The specific method of the tailed-crop screening is the same as the tailed-crop screening method in 2-1).

3) Phenotypic analysis of scoliosis of ccdc57 knockout zebra fish

3-1) appearance feature collection of the knockout zebra fish: wild type and L1 strain zebra fish were anesthetized by soaking in 0.016% tricaine solution and photographed under a microscope. The lateral and dorsal aspects of wild type and two ccdc57 knockout lines of zebrafish are shown in fig. 2: the axis of the wild zebra fish is linear, and the axes of the two knockout strains of zebra fish are S-shaped or wavy.

3-2) knocking out zebra fish bone feature collection: wild type and L1 strain zebra fish are soaked in 0.016% tricaine solution for anesthesia, then placed on ice for 10 minutes for death, soaked in 4% paraformaldehyde solution for fixation for 2 hours, transferred to 100% methanol solution for elution for 10 minutes, and then placed in 100% methanol solution for storage. Samples stored in methanol solution were removed and eluted 2 times for 5 minutes each with PBS solution before Micro CT scanning. The sample is then placed on a stage for scanning. As shown in fig. 3, Micro CT showed skeletal features of wild type and L1 line zebrafish: E-F shows that the bones of the wild zebra fish are linear. G-L shows that bones of zebra fish of the L1 knockout strain are in an s shape or a wave shape, wherein G-H bending degree is light, I-J bending degree is medium, and K-L bending degree is severe.

3-3) microinjection to complement normal ccdc57 mRNA: primers (SEQ ID NO.7 and NO.8) are designed, CDS sequences (Coding sequences and Coding sequences) of wild zebra fish cccd 57 are amplified and connected to a PsP64 vector, in vitro transcription is carried out to synthesize wild cccd 57mRNA, the concentration is measured by a spectrophotometer after purification, and the wild zebra fish cccd is stored in a refrigerator at-80 ℃. Before pre-injection, ccdc57mRNA is taken out, diluted to 100ng/ul, 50ng/ul and 25ng/ul respectively, injected by adopting 3 concentration gradients, the optimal concentration is searched, and finally the concentration of 25ng/ul is determined to have the saving effect and the fatality rate is lower. In the formal refilling injection experiment, 0.1ul 25ng/ul ccdc57mRNA is added into a micro-injection needle and injected into 200 fertilized eggs, wherein 100 fertilized eggs are wild-type fertilized eggs, and the other 100 fertilized eggs are L1 knockout strains. After hatching, the injected and non-injected wild type and L1 strain embryos are soaked in 0.016% tricaine solution for anesthesia, and the embryos are photographed under a body microscope. The results are shown in fig. 4, which shows that injection of the complemented normal ccdc57mRNA can effectively rescue the early stage of embryo axial ventral bending phenotype of the L1 strain, indicating that the scoliosis phenotype of the knockout strain is indeed caused by deletion of the ccdc57 protein.

3-4) detecting the expression change of epinephrine and its receptor by fluorescent quantitative PCR: 72hpf wild type, L1 mutant line and complemented L1 mutant line embryos are collected, total RNA is extracted by a Trizol method, and a cDNA library is further synthesized by reverse transcription. Taking a cDNA library as a template, preparing a PCR system by using primers (SEQ ID NO.9-SEQ ID NO.20) for amplifying specific fragments of gapdh, cccd 57, urp1, urp2, uts2r and uts2ra, SYBR green premixed solution and deionized water, performing PCR amplification and fluorescence signal monitoring by using an ABI fluorescence quantitative PCR instrument, and finally, taking gapdh as an internal reference, and relatively comparing the expression quantity of each target gene of different samples. The results are shown in fig. 5, which shows that the expressions of L1 strain tail pressor peptides 1 and 2 and the receptors thereof, which lack ccdc57 protein, are reduced, and the expressions of L1 strain tail pressor peptides 1 and 2 and the receptors thereof can be obviously improved after excessive normal ccdc57mRNA is supplemented, thus indicating that ccdc57 can regulate body axis development by influencing a tail pressor peptide signal pathway.

It should be noted that, for the convenience of description, corresponding Forward primers and reverse primers of specific fragments of gapdh, cccd 57, urp1, urp2, uts2R and uts2ra, denoted by corresponding fragments and letters F, R, F: Forward primer and R: Reversed primer reverse primer, respectively, are extended. The specific sequence of the forward primer is shown as SEQ ID NO.9 of the sequence table, the specific sequence of the forward primer is shown as gapdh, and the specific sequence of the reverse primer is shown as SEQ ID NO.10 of the sequence table.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Sequence listing

<110> university of Master in Hunan

<120> method for establishing scoliosis animal model

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tgcatcattt cagtctttg 19

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ggcttctcac aaacgaggac 20

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tcaagaaagc agcacgggt 19

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gtttgttcgc tgtggcttgt 20

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gttgaaaaac cggggactgc 20

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acattctggc tgtggtttg 19

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gtccgtcttc aacctctgct ac 22

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agaggaaaca gcaatggacg 20

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tgttggtttt cttggttgac g 21

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caagcatctt taccctcacc g 21

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gggcaaactg agagcgatgg 20

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acccgttcct ctacactttg c 21

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gaggtgaccg ctgaaaacg 19

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ggaggaaagc 10

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ggaggaaagg gacagc 16