Ke's syndrome animal model and application thereof
阅读说明：本技术 克氏综合征动物模型和其应用 (Ke's syndrome animal model and application thereof ) 是由 李卫 刘超 陈子江 刘洪彬 王丽娜 于 2019-04-04 设计创作，主要内容包括：本发明提供了研究克氏综合征的动物模型,所述动物缺失Usp26。所述动物模型是可育的,其可产生XXY染色体组的后代。本发明还提供了所述动物模型在研究克氏综合征中的用途。本发明还提供了所述动物模型的产生方法,包括使得所述动物的内源性Usp26产生突变或缺失,例如通过CRISPR-Cas9系统进行基因编辑。(The invention provides an animal model for studying Klebsiella syndrome, which animal lacks Usp 26. The animal model is fertile, which can produce offspring of the XXY genome. The invention also provides the application of the animal model in the research of the Klebsiella syndrome. The invention also provides a method for producing the animal model, which comprises the step of mutating or deleting endogenous Usp26 of the animal, such as gene editing through a CRISPR-Cas9 system.)
1. A transgenic animal deficient in Usp26 for use in studying crohn's syndrome.
2. The animal of claim 1 which is a male rat or mouse.
3. The animal of claim 1 which is fertile.
4. A method of producing a transgenic animal comprising mutating or deleting endogenous Usp26 of said animal.
5. The method of claim 4, wherein the animal is a male rat or mouse.
6. The method of claim 4, wherein the mutation that results in the non-expression of the USP26 protein is introduced by Usp26 on the X chromosome of the animal.
7. The method of claim 4 wherein the deletion is made by Usp26 on the X chromosome of the animal.
8. Use of a transgenic animal as defined in any of claims 1 to 3 for studying kruse syndrome.
9. The use of claim 8, which comprises producing progeny with said transgenic animal.
10. The use of claim 9, wherein the progeny have the XXY genome.
Technical Field
The present invention relates to the field of molecular biology and animal models for disease research. In particular, the invention relates to a fertile animal model for studying kruse syndrome and methods for producing and using the same.
Background
Kleinefelter's Syndrome (KS) is a disease that severely affects the ability to produce offspring. The Klebsiella syndrome, also known as XXY and 47XXY syndrome, is a disease characterized by a 47XXY karyotype, with two or more X chromosomes in males. The patients with the Klebsiella syndrome inevitably have infertility, the prevalence rate of the Klebsiella syndrome in sterile men is as high as 3% -4%, and the prevalence rate of the Klebsiella syndrome in azoospermia patients is as high as 10% -12%. Several complications of KS have also been reported, including metabolic disturbances, certain psychosocial problems, and susceptibility to the formation of certain tumors. Since the clinical manifestations of patients with crohn's disease may be similar to those of normal men, there is a serious under-diagnosis problem.
Since Harry Klinefelter first described the krebs syndrome in 1942, a number of studies have attempted to reveal the pathogenic mechanisms of KS origin. There is an extra X chromosome in the krebs syndrome, which may be due to the fact that chromosomes do not separate during meiosis I or meiosis II occurring in the maternal ovum, or during meiosis occurring in the paternal spermatozoa. Maternal advanced age is the only evidence-based risk factor for crohn's syndrome, which is also an important cause of other autosomal trisomies. However, this effect is limited to a fraction of cases derived from MI, whereas maternal MII appears to be independent of maternal age. It has been found that the paternal factor in KS appears to be significantly different. In addition, KS is not considered to be genetic, but rather occurs randomly during meiosis. To date, although the krebs syndrome has been discovered and studied for over 70 years, the molecular mechanisms of KS origin have never been truly elucidated.
Since the molecular mechanism of the origin of the kruse syndrome has not been fully elucidated, animal models useful for studying the origin of the kruse syndrome are not mature, and particularly, there is a lack of animal models useful for producing offspring of the kruse syndrome.
Therefore, there is a need in the art for more research on the molecular mechanism of kruse syndrome, and for new animal models for studying kruse syndrome.
Disclosure of Invention
The invention provides an animal model for studying Crohn's syndrome and the use thereof (including progeny thereof) for studying Crohn's syndrome or the risk of having Crohn's syndrome in the progeny. Specifically, the invention firstly determines through research that the mutation of USP26 is the root cause of one of the occurrences of the Klebsiella syndrome, and provides an animal model which can maintain the fertility and can generate the offspring of the Klebsiella syndrome according to the principle.
In particular, the invention provides a transgenic animal for studying kruse syndrome, which lacks Usp 26. In one aspect of the invention, the animal is a male rat or mouse. The invention also provides tissues or cells of the transgenic animal.
USP26(ubiquitin specific peptidase 26), ubiquitin-specific peptidase 26, is one of the members of the ubiquitin-specific processing (UBP) family of peptidases. By radiation hybridization analysis, Wang et al (Nature Genet.27: 422-426,2001) mapped the human Usp26 Gene to the X chromosome (Gene ID: 83844). The Usp26 Gene of the mouse was also mapped to the X chromosome (Gene ID: 83563).
The transgenic animals provided by the invention are fertile. Although the Klebsiella syndrome is a disease that seriously affects the ability to produce offspring, the transgenic animals provided by the present invention are themselves fertile and thus can be stably passaged. Part of the progeny that they produce may have an XXY karyotype, suitable for use in studying kliner's syndrome.
The invention also provides a method for producing the above transgenic animal, which comprises mutating or deleting endogenous Usp26 of the animal. In yet another aspect of the invention, the mutation that results in the non-expression of Usp26 protein is introduced by Usp26 on the X chromosome of said animal. In yet another aspect of the invention, by deleting Usp26 on the X chromosome of the animal,
various methods of altering mammalian genes are known in the art. Including methods of altering the genome of a mammal and allowing said alteration to be transmitted in the offspring of said animal. Gene editing can be performed, for example, by the CRISPR-Cas9 system.
The invention also provides application of the transgenic animal in researching the Klebsiella syndrome. The invention also provides the application of the tissues or cells of the transgenic animal in the research of the Klebsiella syndrome.
In yet another aspect of the invention, the use of the transgenic animal described above for studying kruse syndrome comprises producing offspring using the transgenic rodent. The progeny includes progeny having the XXY genome.
In this context, the protein symbols are not italicized and are all capitalized; the gene symbols are in italics. For example, USP26 is a protein and the gene encoding the protein is written as USP 26. Sometimes, however, italics is not used in the present context for the gene symbols. For example, sometimes "USP 26" or "USP 26 gene" herein denotes the gene USP26 encoding USP26 protein.
Drawings
FIG. 1 shows that Usp 26-deficient mice give rise to 41XXY progeny.
FIG. 1A shows Usp26-/YUSP26 protein is not present in testis. At Usp26+/YAnd Usp26-/YUSP26 immunoblots were performed in testis.
FIGS. 1B and 1C show Usp26-/YThe fertility of mice decreases with age. Usp26 at 2 months of age and 6 months of age+/Y，Usp26-/YFertility assessment experiments were performed in mice (fig. 1B) and their litter size was observed (fig. 1C).
FIG. 1D shows WT and Usp26 mutant alleles.
FIG. 1E shows Usp26-/YGenotyping of mouse offspring.
FIG. 1F shows Usp26 at 2-month old and 6-month old+/Y，Usp26-/YThe proportion of 41XXY mice in the progeny of the mice.
FIG. 1G shows Usp26+/-/YThe mouse had testis smaller than the control group.
FIG. 1H shows Usp26+/YAnd Usp26+/-/YTestis weight/body weight ratio in mice.
FIG. 1I, right panel, shows the passage of hematoxylin and eosin (H)&E) Dyeing pair Usp26+/YAnd Usp26+/-/YThe mouse seminiferous tubules and epididymis cauda were histologically analyzed. The left panel is shown in Usp26+/-/YIn mice, the number of sperm in the tail epididymis was significantly reduced.
FIG. 2 shows that USP26 participates in sex chromosome pairing.
FIG. 2A shows that USP26 is mainly expressed in testis. Immunoblotting of USP26 was performed in heart, liver, spleen, lung, kidney, intestine, brain, ovary and testis.
Figure 2B shows the positioning of USP26 during meiosis. Immunofluorescence analysis of SCP3 (red), USP26 (white) was performed in WT spermatocytes. Nuclei were stained with DAPI (blue).
FIG. 2C is a representation of the sequence shown in Usp26-/YX and Y chromosomes are unpaired in spermatocytes. At Usp26+/YAnd Usp26-/YImmunofluorescence analysis of Chr X-FISH (Green), Chr Y-FISH (Red) and SCP3 (white) in spermatocytes. The arrow indicates the X chromosome.
FIG. 2D shows the quantification of unpaired Chr X and Chr Y.
FIG. 2E shows a representation in Usp26+/YAnd Usp26-/YImmunofluorescence analysis of SCP3 (green), ATR (red) and p-ATM (pink) was performed in spermatocytes. Nuclei were stained with DAPI (blue). Arrows indicate sex chromosomes.
FIG. 3 shows that Usp26 deficient mice produce XY aneuploid sperm.
FIGS. 3A and 3B are shown in Usp26-/YLagging chromosomes were observed in metaphase I of spermatocytes. At Usp26+/YAnd Usp26-/YImmunofluorescence analysis of tubulin (green) in spermatocytes was performed. Nuclei were stained with DAPI (blue). Arrows indicate lagging chromosomes.
FIG. 3C is a representation of the sequence shown in Usp26+/YAnd Usp26-/YProportion of metaphase I spermatocytes exhibiting lagging chromosomes in mice.
Fig. 3D shows that Usp26 deficient mice produced XY aneuploid sperm. At Usp26+/YAnd Usp26-/YChr X (Green), Chr Y (Red) FIS in spermAnd H, measuring. Nuclei were stained with DAPI (blue). Arrows indicate the Y chromosome and arrows indicate the X chromosome.
FIG. 3E shows Usp26 that was older at 2 months and 6 months+/YAnd Usp26-/YQuantification of different types of sperm in mice.
FIG. 4 shows that non-segregation at MI results in sex chromosome aneuploid sperm.
FIG. 5 shows that disruption of Usp26 has no effect on follicular development and chromosome segregation in female mice.
FIG. 5A shows Usp26+/+And Usp26-/-Hematoxylin and eosin (H) of ovary in mice&E) And (6) dyeing.
FIG. 5B is shown at Usp26+/+And Usp26-/-Immunofluorescence analysis of Tub (green) in oocytes was performed. Nuclei were stained with DAPI (blue).
FIG. 6 shows that defects in Usp26 lead to pachytene and meiotic arrest.
Usp26+/YAnd Usp26-/YRepresentative TUNEL produced in testis. Staining with TUNEL (Green) and DAPI (blue) from Usp26+/YAnd Usp26-/YParaffin sections of testis to show dead cells in stage IV and XII tubules with pachytene and mesospermic cells, respectively. The arrow tip represents a lagging chromosome.
FIG. 7 shows that unpaired sex chromosomes can also be detected in other types of Usp 26-deficient mice.
Fig. 7A shows that the X and Y chromosomes are unpaired in two types of Usp26 deficient mouse spermatocytes. Immunofluorescence analysis of SCP3 (green), γ H2AX (red) was performed in WT and Usp26 deficient spermatocytes. Nuclei were stained with DAPI (blue).
FIG. 7B shows immunofluorescence analysis of SCP3 (green), TRF1 (red) and ATR (white) in WT and Usp26 deficient spermatocytes. Nuclei were stained with DAPI (blue).
Detailed Description
The spirit and advantages of the present invention will be further illustrated by the following examples, which are provided by way of illustration and are not intended to be limiting.