阅读说明：本技术 一种突变基因及其用于构建蹼足病小型猪模型的用途 (Mutant gene and application thereof in constructing small pig model with webbed foot disease ) 是由 周琪 赵建国 海棠 贾启涛 曹春伟 王红梅 张颖 郑千涛 王霄 于 2018-08-21 设计创作，主要内容包括：本发明提供一种小型猪的RIPK4突变基因,所述突变基因与野生型猪RIPK4基因相比,具有c.1397_1398insA的突变。本发明提供了一种构建体,所述构建体包含所述RIPK4突变基因。本发明还提供了一种重组细胞,所述重组细胞由所述构建体转化受体细胞获得。本发明提供了一种制备蹼足病小型猪模型的方法,所述方法包括：改变小型猪的RIPK4基因,使RIPK4基因的位于第8号外显子的第1397位碱基和1398位碱基之间插入腺嘌呤A。利用本发明提供的RIPK4突变基因制备大型蹼足病猪模型,研究蹼足病的发病机理,对于临床预防、诊断和治疗蹼足病具有重大的指导意义。(The invention provides a miniature pig RIPK4 mutant gene, which has c.1397_1398insA mutation compared with the wild pig RIPK4 gene. The invention provides a construct, which comprises the RIPK4 mutant gene. The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. The invention provides a method for preparing a small pig model with webbed foot disease, which comprises the following steps: the RIPK4 gene of the miniature pig is changed, so that adenine A is inserted between 1397 th base and 1398 th base of the RIPK4 gene which is positioned in the No. 8 exon. The RIPK4 mutant gene provided by the invention is used for preparing a large-scale webbed-foot disease pig model, the pathogenesis of the webbed-foot disease is researched, and the invention has great guiding significance for clinical prevention, diagnosis and treatment of the webbed-foot disease.)

1. A minipig RIPK4 mutant gene having a mutation of c.1397 — 1398insA compared to the wild-type pig RIPK4 gene;

preferably, the sequence of the RIPK4 mutant gene is shown as SEQ ID NO. 29;

preferably, the miniature pig is a bama miniature pig.

2. A construct comprising the RIPK4 mutant gene of claim 1.

3. A recombinant cell obtained by transforming a recipient cell with the construct of claim 2;

preferably, the recombinant cell is a porcine cell, more preferably a bama miniature porcine cell.

4. A method of making a mini pig model of webbed foot disease, the method comprising:

the RIPK4 gene of the miniature pig is changed, so that adenine A is inserted between 1397 th base and 1398 th base of the RIPK4 gene which is positioned in the No. 8 exon.

5. A method for screening a minipig model with a mutated RIPK4 gene for webbed foot disease, the method comprising the steps of:

1) extracting nucleic acid DNA of a biological sample to be detected;

2) determining the sequence of the nucleic acid DNA;

3) a sequence of the nucleic acid, or a complement thereof, having a c.1397 — 1398insA mutation compared to the wild-type RIPK4 gene, the mutation being indicative of webbed foot disease;

the biological sample is selected from at least one of blood, skin, hair, and muscle.

6. The method according to claim 5, wherein in step 2), determining the sequence of the nucleic acid comprises the steps of:

carrying out PCR by using a specific primer of a pig RIPK4 gene to obtain an amplification product and sequencing the amplification product;

preferably, the sequence of the forward primer is shown as SEQ ID NO. 27, and the sequence of the reverse primer is shown as SEQ ID NO. 28.

7. A kit for screening a small pig model with RIPK4 gene mutation webbed foot disease is characterized by comprising specific primers of RIPK4 gene.

8. Use of the mutant gene of claim 1, the construct of claim 2, the recombinant cell of claim 3, or the kit of claim 7 in the preparation of an animal model for screening for the treatment and/or prevention of webfoot disease; preferably, the animal model is a mammalian model; more preferably, the mammal is a mouse, monkey or miniature pig.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an RIPK4 mutant gene, application of the mutant gene in constructing a small pig model with webbed foot disease (Bartscoas-Papas syndrome) and a construction method of the small pig model with the webbed foot disease.

Background

Miniature pigs are a very good animal model for studying the etiology, molecular mechanisms and treatment of human diseases. The Bama miniature pig is a local pig breed special for China, is mainly produced in Guangxi province of China, and is characterized by typical 'two-head black' appearance, namely, hairs on the head and the tail of the hip are black, and the other parts are white. In recent years, the Bama pigs have the advantages of small size and similar anatomical structure to human, and are gradually considered as good materials for constructing large animal models for human disease medical research by the scientists, so the Bama pigs have extremely high breeding value.

Webbed-foot disease (bartscas-pasas syndrome) is a low-morbidity hereditary birth defect in newborns, often leading to death, mainly manifested as cheilosis/cleft palate symptoms, but also other abnormalities, such as skin folds, merging toes (syndactyly), genital abnormalities, etc.

The existing Bartsocas-Papas syndrome disease model is a mouse, but the physiological structure of the mouse is far away from that of a human, so that the disease model needs to be constructed in a large animal similar to the physiological structure of the human, and support is provided for pathological research, drug screening, pharmacodynamic evaluation and other researches aiming at the disease.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the RIPK4 mutant gene of the miniature pig, the phenotype and the genetic pattern of the transgenic family miniature pig with the mutant gene are consistent with human webbed foot disease (Bartsocas-Papas syndrome), and the mutant gene can be used as a large animal model of the human genetic disease, thereby providing support for pathological research, drug screening, drug effect evaluation and other researches aiming at the disease.

In one aspect, the invention provides a minipig RIPK4 mutant gene having a mutation of c.1397 — 1398insA compared to the wild-type pig RIPK4 gene.

Specifically, the mutation site of the mutant gene is located in the No. 8 exon, and the adenine (A) is inserted between 1397 base and 1398 base, so that the code shift mutation of the coded amino acid occurs (p.Asn466LysfsTer169). Specifically, the 466-position asparagine (Asn) of the RIPK4 mutant gene is mutated into lysine (Lys), and the later translated amino acid is subjected to frame shift mutation.

Preferably, the RIPK4 mutant gene sequence is shown as SEQ ID NO. 29.

Preferably, the miniature pig is a bama miniature pig.

In another aspect, the invention provides a construct comprising the RIPK4 mutant gene.

The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. Preferably, the recombinant cell is a porcine cell, more preferably a bama miniature porcine cell.

According to the embodiment of the invention, the genome of the recombinant animal cell has a nucleic acid sequence coding a mutant RIPK4, wherein compared with a wild-type RIPK4, the protein of the mutant RIPK4 has mutation of asparagine (Asn) at position 466 in translation to lysine (Lys) and the later translated amino acid has frame shift mutation.

In yet another aspect, the present invention provides a method of preparing a small pig model for webbed foot disease, the method comprising:

the RIPK4 gene of the miniature pig is changed, so that adenine A is inserted between 1397 th base and 1398 th base of the RIPK4 gene which is positioned in the No. 8 exon.

In some embodiments of the invention, according to actual needs, by using genetic engineering technology, the RIPK4 gene of a normal pig is changed, adenine A is inserted between the 1397 th base and the 1398 th base, and a flipper-foot-affected pig model is obtained. In other embodiments, genetic engineering techniques may be used to alter the corresponding sites of RIPK4 gene in animals other than pigs to obtain the desired large or small animal models of webbed foot disease, such as a monkey model of webbed foot disease, a mouse model of webbed foot disease, and the like.

The invention also provides a method for screening the small pig model with the webbed foot disease of RIPK4 gene mutation, which comprises the following steps:

1) extracting nucleic acid DNA of a biological sample to be detected;

2) determining the sequence of the nucleic acid DNA;

3) a sequence of the nucleic acid, or a complement thereof, having a c.1397 — 1398insA mutation compared to the wild-type RIPK4 gene, the mutation being indicative of webbed foot disease;

the biological sample is selected from at least one of blood, skin, hair, and muscle.

In step 2), determining the sequence of the nucleic acid comprises the steps of:

carrying out PCR by using a specific primer of a pig RIPK4 gene to obtain an amplification product and sequencing the amplification product;

preferably, the sequence of the forward primer (F) is shown as SEQ ID NO:27, and the sequence of the reverse primer (R) is shown as SEQ ID NO: 28.

SEQ ID NO:27 5’-3’:CGCCCCTCTAGGTCTCAGAT；

SEQ ID NO:28 5’-3’:GGCATTGACGCTGATCTTGC

The invention also provides a kit for screening the RIPK4 gene mutation flipper foot disease miniature pig model, which comprises a liquid or powdered specific primer of the pig RIPK4 gene. The kit may include other reagents required for PCR, such as buffers, dntps, polymerase; reagents and consumables required for recovering PCR products, such as a sol solution, a collection tube, a washing solution and the like, can also be included. The DNA of a sample to be detected is used as a template, the kit and the method for screening the RIPK4 gene mutation webbed-foot sick pig are used for detection, the operation is simple and convenient, and a large number of samples can be rapidly identified.

In order to obtain a large animal model with webbed foot disease, ENU (N-ethyl-N-nitrosourea) chemical mutagenesis is carried out on the Bama miniature pig, and ENU is injected into a wild male miniature pig to obtain an F0 generation miniature pig; mating the miniature pig with a wild type female miniature pig of the same breed to obtain an F1 generation miniature pig; mating the F1 generation male miniature pig with a wild type female miniature pig to obtain an F2 generation miniature pig; the F1 generation miniature pig male miniature pig and the F2 generation miniature pig mate to obtain an F3 generation miniature pig (51 wild boars, 34 wild sows, 12 deformed boars and 9 deformed sows, wherein the deformed proportion is 21/106-19.81%), the F3 generation miniature pig is subjected to phenotype screening to obtain a family miniature pig with a webbed-foot disease phenotype, and the family miniature pig carries a RIPK4 homozygous mutant gene.

The webbed podopathy-like inheritance pattern of the family miniature pig conforms to Mendelian inheritance law of autosomal monogenic recessive inheritance (51 normal phenotype boars, 34 normal phenotype sows, 12 malformed boars and 9 malformed sows, wherein the malformation proportion is 21/106-19.81%). The number of wild-type individuals and mutant phenotype individuals in the F3 generation were counted and aligned, since wild-type: the mutant ratio is about 1:1, and the separation law of Mendelian dominant inheritance 1:1 is met; also, the ratio of existing females to males in the mutant was close to 1:1, indicating that the mutant phenotype is independent of sex, thereby determining that the mutant phenotype is autosomal dominant.

In one embodiment according to the present invention, the injected dose of the ENU is 50 to 100mg/kg, more preferably, the injected dose of the ENU is 60 to 70 mg/kg; further preferably, the injection dose of the ENU is 65 mg/kg.

In one embodiment according to the present invention, further comprising: and (3) detecting the semen quality of the wild type male miniature pig after ENU injection, and mating the wild type male miniature pig after injection with the wild type female miniature pig of the same variety after the semen quality of the wild type male miniature pig after injection returns to a normal level.

The phenotypic analysis result shows that the small pig model with the web-foot disease has the advantages of obviously reduced body type, black spots on the back skin, even complete blackening, adhesion of ears on the skin, fusion of mouth and nose, no tail, short limbs and incomplete toe development.

Through whole genome correlation analysis, the pathogenic gene of the small pig model with the webbed foot disease is determined to be RIPK4 gene, and the exon of RIPK4 gene is completely sequenced, so that the mutation site of the mutant gene is positioned between No. 8 exon, 1397 base and 1398 base, adenine A is inserted, the 466 asparagine (Asn) in protein translation of RIPK4 can be mutated into lysine (Lys), and the later translated amino acid has frame shift mutation. According to data retrieval, the RIPK4 gene is well conserved in mammals such as human beings, monkeys, pigs, cattle, sheep, horses, cats, dogs, rabbits, mice, rats and the like, so that the RIPK4 mutant gene provided by the invention is used for preparing a large-scale webfoot disease pig model, researching the pathogenesis of the webfoot disease and has great guiding significance for clinical prevention, diagnosis and treatment of the webfoot disease.

In still another aspect, the present invention provides the use of the genetically mutant animal of the present invention as a model for studying webbed foot disease in humans. Preferably, the genetically mutant animal is a bama minipig.

In one embodiment according to the invention, the invention determines the mutation position by performing genetic linkage analysis and gene clone sequencing on a miniature pig model; preferably, the mutation site closely linked to RIPK4 site is obtained by gene cloning sequencing.

In a further aspect, the present invention provides the use of the mutant gene, construct, recombinant cell or kit of the present invention in the preparation of an animal model for screening for the treatment and/or prevention of webbed foot disease; preferably, the animal model is a mammalian model; more preferably, the mammal is a mouse, monkey or miniature pig.

The phenotype of the miniature pig with the RIPK4 gene mutation is very similar to human webbed foot disease, so that a very good animal model of the human webbed foot disease is provided, and the miniature pig with the RIPK4 gene mutation can be used for pathological research, treatment mode research and drug screening of the human webbed foot disease. In addition, the method provided by the invention avoids hybridization of the Bama pigs and other white pig breeds, does not introduce exogenous genetic information, and thus does not increase the complexity of the genetic background of the Bama pigs, which is beneficial to the establishment of the next purification genetic background and inbred line.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a whole body phenotype of a Bama miniature pig model (Mut) and a wild type piglet (WT) of the present invention;

FIG. 2 is the genotyping results of the Bama minipig model (Mut) and wild type piglets (WT) of the webfoot disease of the present invention, with the mutation sites of the coding regions of the genes indicated by arrows;

FIG. 3 shows the skin of a small pig with a webbed foot disease of the present invention and the skin of a wild type pig are HE-stained, wherein WT is the wild type pig and Mut is the small pig with a webbed foot disease of the present invention, and as a result, the horned layer of the epidermis of the mutant pig is found to be remarkably abnormal, mainly as the horned layer is more loose;

FIG. 4 shows the expression of RIPK4 protein of a small pig model with webbed foot disease and a wild type piglet, the inventors constructed in vitro overexpression vectors RIPK4-HA-WT and RIPK4-HA-Mut and transfected 293T cells, and then detected the expression of RIPK 4; the results show that the protein is significantly degraded after RIPK4 mutation, which indicates that the mutation affects the stability of the protein.

Detailed Description

The present invention is further described below in conjunction with the following figures and examples, it being understood that the examples are intended to further illustrate and explain the present invention, and are not intended to limit the present invention.

The Guangxi Bama miniature pig is purchased from third army medical university and bred in the northern big animal research base of animal research institute of Chinese academy of sciences

ENU Sigma(N8509bulk package)

Anesthetic (ketamine) northeast university of agriculture

Jiangxi Baokeming injection Jiangxi Boda animal medicine health-care company