CN113999874B - Construction method and application of PHB1 gene knock-out non-human animal - Google Patents

Abstract

The invention relates to a construction method of a PHB1 gene knock-out non-human animal and application thereof in the field of toxicology and biological medicine, wherein a Cre/LoxP and CRISPR/Cas9 system is utilized to obtain the non-human animal, the PHB1 protein which is not expressed or expressed in PHB1 protein in choroid plexus tissues of the non-human animal has no function, can be used as an animal model for screening obesity, diabetes, choroid plexus related diseases, tumors or inflammatory drugs and researching the choroid plexus toxicology by heavy metals or other toxic substances, and has important application value for researching and developing the obesity, the diabetes, the choroid plexus related diseases, the tumors or the inflammatory drugs.

Description

Construction method and application of PHB1 gene knock-out non-human animal

Technical Field

The application relates to a construction method and application of a gene-knocked non-human animal, in particular to a construction method based on PHB1 gene brain-knocked non-human animal and application thereof in the fields of toxicology and biomedicine.

Background

Choroid Plexus (CP) is formed by the division of the ependyma into the ventricles of the brain after invagination, and is arranged in a net shape in the ventricles of the brain, and can be divided into three types, namely, a lateral ventricular choroid plexus, a third ventricular choroid plexus and a fourth ventricular choroid plexus according to different anatomical locations. The choroid plexus is not only an important source of cerebrospinal fluid (CSF), but also can selectively transport some blood-borne substances into CSF through the blood-cerebrospinal fluid barrier (BCB) formed by its active epithelium, so as to maintain the homeostasis of CSF environment, while also preventing possible damage to brain tissue by some harmful factors present in the peripheral circulation.

In the toxic action and pharmacological action of exogenous substances, the choroid plexus has double characteristics, namely the nerve protection effect (preventing toxicant from entering brain tissues and cerebrospinal fluid); and some target tissues of neurotoxins (as sites of toxicant accumulation; or the toxic effects of exogenous substances can directly lead to structural and functional impairment of BCB). From the perspective of neuroprotection, the choroid plexus has a large number of microvilli and specific functional protein receptors/transporters, can selectively and actively absorb or discharge some specific substances, can be used as storage sites for some toxic heavy metals (such as compounds of lead, mercury, cadmium and the like) and other exogenous compounds (such as drugs and the like), shields and concentrates the toxic heavy metals and toxic substances in choroid plexus tissues, prevents the toxic heavy metals and toxic substances from entering cerebrospinal fluid, keeps the stability of cerebrospinal fluid components, and further plays a role in protecting the central nervous system.

The choroid plexus also plays an important role in the development, maturation, aging process, endocrine regulation of the brain, and the pathogenesis of some neurodegenerative diseases. In addition, studies of choroid plexus in brain tissue inflammation, aging, and secondary neurotoxic mechanisms have been reported. However, domestic studies on these aspects are limited, and focus mainly on choroid plexus cysts and tumors, and further emphasis has not been placed on study of choroid plexus-related toxicology and pharmacology. Through systematic research on the reaction mechanism of BCB under the action of internal and external compounds, the action exerted in the process of repairing nerve injury and after injury, the regulation mechanism and the like, it is possible to discover some nerve toxicants \ early diagnosis markers of nerve injury, transport proteins of CNS (central nervous system) specific drugs and the like, and promote the development of the diagnosis and treatment level of CNS diseases.

Inhibin protein (PHB 1) is composed of 275 amino acids, and is a protein which is highly conserved in structure and widely distributed in mitochondria of eukaryotes and has various functions. It is mainly involved in maintaining mitochondrial structure and function, protecting cells against aging, regulating intracellular iron homeostasis, and participating in mitochondrial protein stabilization and mitochondrial structure maintenance as a chaperone protein. Furthermore, the distribution of PHB1 in the nucleus and cytoplasm makes it involved in cell cycle, apoptosis and transcriptional regulation, and as a cell surface receptor in intracellular signal transduction. The expression change of PHB1 directly affects the increase of oxidative stress and the dysfunction and structural abnormality of mitochondria, which is closely related to the induction of certain diseases such as cancer, inflammatory diseases, insulin resistance type 2 diabetes and the like and the drug treatment of chronic diseases.

The role of PHB1 in mitochondria is the most deeply and clearly researched at present, PHB1 and PHB2 are combined with each other to form foreign body dimers, each 12-16 foreign body dimers are further polymerized to form concentric circular ground-like structures, and are anchored on the inner membrane of mitochondria through the common amino-terminal lipophilic domain of the foreign body dimers, play the role of molecular chaperone protein, maintain the sum function of mitochondria, and serve as iron storage protein in mitochondria to maintain the iron dynamic balance in vivo and participate in anti-apoptosis.

In recent years, it has been found through immunohistochemistry and co-immunoprecipitation that PHB1 colocalizes with other tumor growth inhibitory proteins such as E2F transcription factor family member, Rb protein family member, p53 tumor inhibitory protein and the like in the nucleus of the cell in breast cancer cell lines and prostate cell lines, and exerts cell proliferation inhibition and pro-apoptosis effects through their respective interactions. This is also the reason why PHB1 is gaining attention as a target protein for the research of antitumor drugs.

Krishnaraaj and Thomas established PHB1 gene silencing cell line by siRNA technology in 2005, found another role of PHB1 protein, namely Ras activation and Ras-1 binding, which are involved as chaperones of Raf-1 on plasma membrane and anchoring firmly to plasma membrane, occurred a series of phosphorylation processes, such as tyrosine 341 and Serine 338 (tyrosine 341, Serine 338), dephosphorylation (Serine 259), dimerization reaction, etc. to further activate Raf-1, and exerted the role of regulating processes of cell proliferation, differentiation, cell death, mitosis, migration, inflammation, etc. by phosphorylating MEK protein kinase, ERK kinase (the Serine/thionine specific extracellular signal-regulated kinase, ERK) and MAPK kinase (mitogen-activated protein kinase, MAPK).

In addition to its cell cycle regulating effect, PHB1 has multiple functions, and its different localization and differential expression in various diseases suggest that PHB1 may be involved in the process of disease development and progression. Currently, PHB1 is considered as a potential biomarker index for evaluating the course of disease progression and disease risk prediction of diseases such as liver injury, nasopharyngeal carcinoma and the like, and is used as a new drug target protein for treating diseases such as tumor, obesity, diabetes and the like.

Previous studies have demonstrated that: manganese (6 mg/kg. BW) has pathological damage effect on rat choroid plexus tissue and can cause the expression of PHB1 protein in the choroid plexus tissue to be up-regulated; in vitro studies, under the action of manganese, the choroid plexus epithelial cell line Z310 generates cell cycle block and the expression of PHB1 protein is up-regulated, and the molecular regulation mechanism of PHB1 is combined to conclude that the cell cycle block plays an important regulation role in Z310 cell cycle block caused by manganese, but the exact regulation mechanism is not clear.

Based on the research results and problems and a conditional gene knockout Cre/loxP recombination system, a CRISPR/Cas9 system is applied, a LoxP gene is inserted into introns at the 5 'end and the 3' end of a rat PHB1 gene in a fixed point mode, then the gene sequence constructed in vitro is transferred into a fertilized egg, the fertilized egg replaces the original gene sequence in a cell genome through homologous recombination, the fertilized egg is implanted into the uterus of a pseudopregnant rat to be regenerated into a complete embryo, finally, a transgenic rat with loxP sites in introns at two sides of a target gene is formed, the Adgra3-CreERT2 gene is knocked into the rat to be hybridized with the rat, and then the PHB1 gene brain choroid plexus tissue conditional knockout rat can be screened out from offspring. The conditional knockdown of the PHB1 gene by a non-human animal construction is helpful for verifying and mining a definite molecular regulation mechanism of PHB1 in the apoptosis and cycle block effects of manganese-induced choroid plexus tissue epithelial cells from the whole animal level, and how the consequences of the manganese-induced choroid plexus epithelial cell cycle block regulated by PHB1 influence the secretion function of choroid plexus, influence the synthesis and secretion of dopamine neuron-specific neurotrophic factor GDF-15 and influence the damage repair of dopamine neurons finally, so that the development of a non-human animal model for the PHB1 gene is urgently needed in the art.

Non-patent document "Liver-specific deletion of suppression 1 residues in specific Liver deficiency in jury, fibrosis, and hepatocellular cancer in mice" Hepatology, 2010, 52(6): 2096-.

Therefore, the prior art discloses only liver PHB1 gene knockout mice for liver studies, but does not disclose choroid plexus PHB1 gene knockout non-human animals, and does not disclose PHB1 gene knockout non-human animals constructed using a combination of Cre/loxP recombination system and CRISPR/Cas9 system.

Disclosure of Invention

The invention relates to a PHB1 gene knock-out construction method of a non-human animal, which comprises knocking out a PHB1 gene, so that the PHB1 protein in choroid plexus tissues of the non-human animal is not expressed or the expressed PHB1 protein does not have functions. Preferably, the coding sequence of the PHB1 gene, the genomic DNA sequence, the sequence controlling expression of the PHB1 protein, or the functional region of the PHB1 protein is knocked out.

Preferably, the choroid plexus tissue includes, but is not limited to, epithelial tissue and connective tissue, including, but not limited to, epithelial cells, ependymal cells, macrophages, and the like.

Wherein, the coding sequence of the knocked-out PHB1 gene comprises the coding sequence of exons 4 to 5 of the PHB1 gene.

The invention uses gene editing technology to carry out PHB1 gene knock-out unless the construction of human animal, the gene editing technology includes gene targeting technology using embryonic stem cell, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease, RNAi technology, Cre/LoxP technology, Flp/Frt technology or other molecular biology technology or their combination. Further preferably, the gene editing technology utilizes a combination of CRISPR/Cas9 technology and/or Cre/loxP technology.

Preferably, a homologous specific recombination site recognition sequence is inserted into a non-human animal PHB1 locus to obtain a conditional PHB1 gene knockout Flox non-human animal, a recombinase or a recombinase expression sequence is introduced into the conditional PHB1 gene knockout Flox non-human animal, and the conditional PHB1 gene knockout Flox non-human animal PHB1 gene is deleted to obtain a PHB1 gene knockout non-human animal.

Further preferably, the insertion sites are intron3 and intron 5.

Further preferably, the PHB1 gene knocks out exon4 to exon 5.

Further preferably, the homologated specific recombination site recognition sequences include, but are not limited to LoxP or Frt.

Further preferably, the recombinase includes, but is not limited to Cre or Flp.

More preferably, a LoxP or Frt sequence is inserted into each of intron3 and intron5 of PHB1 gene of the non-human animal to obtain a conditional PHB1 gene knockout Flox non-human animal, the conditional PHB1 gene knockout Flox non-human animal is mated with a non-human animal expressing Cre or Flp recombinase in choroid plexus tissue, and the PHB1 gene knockout non-human animal is obtained by using a Cre-LoxP or Flp-Frt gene recombination system.

Preferably, the non-human animal expressing Cre or Flp recombinase in choroid plexus tissue is obtained by knocking a nucleotide sequence encoding Cre into a gene encoding a protein expressed from the tissue.

Further preferably, the nucleotide sequence encoding Cre can be a nucleotide sequence encoding Cre-ER and a sequence optimized from a protein perspective or a gene perspective, including but not limited to a nucleotide sequence encoding Cre-ERT, Cre-ERT2 or iCre-ERT 2.

Further preferably, the gene encoding the protein expressed from the tissues in the choroid is the Adgra3 gene. The knock-in position is preferably after the promoter, so that the knock-in Cre-encoding nucleotide sequence is under the control of the regulatory elements of the Adgra3 gene.

Preferably, the construction method comprises inserting a LoxP or Frt sequence into the PHB1 locus using sgRNA and/or targeting vectors.

Preferably, the sgRNA 3' end comprises a PAM sequence, and further preferably, the PAM sequence may be AGG, TGG or GGG.

Further preferably, the target site sequence at the 5 'end of the sgRNA target is shown in any one of SEQ ID No.1-7, and the target site sequence at the 3' end of the sgRNA target is shown in any one of SEQ ID No. 8-14.

Still more preferably, the sequence of the target site at the 5 'end of the sgRNA target is shown in SEQ ID No.5, and the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID No. 8.

Preferably, the bottom strand oligonucleotide of the sgRNA has more than 80% homology with any one of SEQ ID nos. 15 to 28 or comprises a nucleotide sequence set forth in any one of SEQ ID nos. 15 to 28.

Further preferably, the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a homonymous specific recombination site recognition sequence, a PHB1 gene nucleotide sequence and a homonymous specific recombination site recognition sequence from 5 'to 3'.

Still more preferably, the nucleotide sequence of PHB1 gene comprises exon4 to exon 5 of PHB1 gene.

Still more preferably, the 5' arm is as shown in SEQ ID NO. 51.

Still more preferably, the 3' arm is as shown in SEQ ID NO. 52.

Still more preferably, the homotropic specific recombination site recognition sequence is LoxP or Frt. The non-human animal of the invention is selected from rodent, pig, chicken, rabbit, monkey and any other non-human animal which can be subjected to gene editing to prepare gene humanization.

Preferably, the non-human animal is a non-human mammal, more preferably, the non-human mammal is a rodent, and even more preferably, the rodent is a rat or a mouse.

In a specific embodiment of the invention, the construction method comprises the steps of introducing a targeting vector, sgRNA targeting PHB1 gene and Cas9 into a non-human animal cell, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the cell to develop, hybridizing offspring with the non-human animal expressing Cre or Flp recombinase, and identifying and screening to obtain the PHB1 gene knock-out non-human animal.

Preferably, the construction method comprises the following steps:

1) preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence from any one or more sgRNA target sequences with sequences shown as SEQ ID NO. 1-14;

2) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, sequentially carrying out enzyme digestion on the fragments to be connected to a framework vector, and carrying out sequencing verification to obtain a pT7-sgRNA vector;

3) respectively synthesizing the forward oligonucleotide and the reverse oligonucleotide in the step 1), and denaturing and annealing the synthesized sgRNA oligonucleotides to form a double strand which can be connected into the pT7-sgRNA vector in the step 2);

4) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step 3) with pT7-sgRNA vectors, and screening to obtain sgRNA vectors.

5) Designing a targeting vector, and constructing and obtaining the targeting vector containing a LoxP/Frt recombination system, wherein the targeting vector contains a PHB1 gene homologous sequence, 2 LoxP/Frt sequences, a3 'arm and a 5' arm;

6) mixing an in-vitro transcription product of the sgRNA vector, Cas9 mRNA and a targeting vector to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of a fertilized egg of the non-human animal, transferring the fertilized egg after injection into a culture solution for culture, and then transplanting the fertilized egg into an oviduct of the non-human animal of a receptor parent for development to obtain an F0 generation non-human animal;

7) checking the F0 generation non-human animal by using a PCR technology, verifying the introduction condition of the targeting vector, and obtaining a positive non-human animal introduced with the targeting vector;

8) crossing the positive non-human animal screened in the step 7) with a non-human animal expressing Cre or Flp recombinase to establish a stable PHB1 gene knock-out non-human animal.

In a second aspect, the present invention relates to a PHB1 gene knock-out non-human animal or its progeny, which is obtained by the above-described construction method.

The third aspect of the invention relates to a construction method of a conditional PHB1 gene knockout Flox non-human animal, wherein a homodromous specific recombination site recognition sequence is inserted into a PHB1 gene locus of the non-human animal.

Preferably, the homologous specific recombination site recognition sequences include, but are not limited to LoxP or Frt.

Preferably, the insertion sites are intron3 and intron5 of PHB1 gene.

In a specific embodiment of the invention, the construction method comprises the step of inserting a LoxP or Frt sequence into an intron3 and an intron5 of a PHB1 gene of a non-human animal respectively to obtain a conditional PHB1 gene knockout Flox non-human animal.

Preferably, the PHB1 gene knocks out exons 4 to 5.

Preferably, the construction method comprises inserting a LoxP or Frt sequence into the PHB1 locus using sgRNA and/or targeting vectors.

Preferably, the sgRNA 3' end comprises a PAM sequence, and further preferably, the PAM sequence may be AGG, TGG or GGG.

Further preferably, the target site sequence at the 5 'end of the sgRNA target is shown in any one of SEQ ID No.1-7, and the target site sequence at the 3' end of the sgRNA target is shown in any one of SEQ ID No. 8-14.

Still more preferably, the sequence of the target site at the 5 'end of the sgRNA target is shown in SEQ ID No.5, and the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID No. 8.

Preferably, the bottom strand oligonucleotide of the sgRNA has more than 80% homology with any one of SEQ ID nos. 15 to 28 or comprises a nucleotide sequence set forth in any one of SEQ ID nos. 15 to 28.

Further preferably, the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a homonymous specific recombination site recognition sequence, a PHB1 gene nucleotide sequence and a homonymous specific recombination site recognition sequence from 5 'to 3'.

Still more preferably, the nucleotide sequence of PHB1 gene comprises exon4 to exon 5 of PHB1 gene.

Still more preferably, the 5' arm is as shown in SEQ ID NO. 51.

Still more preferably, the 3' arm is as shown in SEQ ID NO. 52.

Still more preferably, the homotropic specific recombination site recognition sequence is LoxP or Frt.

The non-human animal of the invention is selected from rodent, pig, chicken, rabbit, monkey and any other non-human animal which can be subjected to gene editing to prepare gene humanization.

Preferably, the non-human animal is a non-human mammal, more preferably, the non-human mammal is a rodent, and even more preferably, the rodent is a rat or a mouse.

In one embodiment of the present invention, the construction method comprises the following steps: constructing a vector for generating the sgRNA and obtaining an sgRNA in-vitro transcription product, constructing a targeting vector containing an exogenous nucleotide sequence, introducing the sgRNA in-vitro transcription product, Cas9 mRNA and the targeting vector into a non-human animal cell, and further implanting into the uterus of a pseudopregnant mother mouse to obtain a non-human animal or a progeny thereof. The non-human animal cell may be a fertilized egg cell, an ES cell or a somatic cell of a non-human animal.

Preferably, the construction method comprises the following steps:

1) preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence from any one or more sgRNA target sequences with sequences shown as SEQ ID NO. 1-14;

2) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, sequentially carrying out enzyme digestion on the fragments to be connected to a framework vector, and carrying out sequencing verification to obtain a pT7-sgRNA vector;

3) respectively synthesizing the forward oligonucleotide and the reverse oligonucleotide in the step 1), and denaturing and annealing the synthesized sgRNA oligonucleotides to form a double strand which can be connected into the pT7-sgRNA vector in the step 2);

4) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step 3) with pT7-sgRNA vectors, and screening to obtain sgRNA vectors.

5) Designing a targeting vector, and constructing and obtaining the targeting vector containing a LoxP/Frt recombination system, wherein the targeting vector contains a PHB1 gene homologous sequence, 2 LoxP/Frt sequences, a3 'arm and a 5' arm;

6) mixing an in-vitro transcription product of the sgRNA vector, Cas9 mRNA and a targeting vector to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of a fertilized egg of the non-human animal, transferring the fertilized egg after injection into a culture solution for culture, and then transplanting the fertilized egg into an oviduct of the non-human animal of a receptor parent for development to obtain an F0 generation non-human animal;

7) and (3) checking the F0 generation non-human animal by using a PCR technology, verifying the introduction condition of the targeting vector, and obtaining a positive non-human animal of the introduction targeting vector, namely the conditional PHB1 gene knockout Flox non-human animal.

The third method of the invention relates to a conditional PHB1 gene knockout Flox non-human animal obtained by adopting the construction method.

In a fourth aspect, the invention relates to a conditional PHB1 gene knockout, Flox, non-human animal as described above for use in knocking in the PHB1 gene of a non-human animal.

The fifth aspect of the invention relates to a sgRNA targeting a PHB1 gene, wherein a target site sequence at the 5 'end of the sgRNA targeting is shown in any one of SEQ ID No.1-7, and a target site sequence at the 3' end of the sgRNA targeting is shown in any one of SEQ ID No. 8-14.

Still more preferably, the sequence of the target site at the 5 'end of the sgRNA target is shown in SEQ ID No.5, and the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID No. 8.

Preferably, the bottom strand oligonucleotide sequence of the sgRNA has more than 80% homology with any one of SEQ ID nos. 15 to 28 or comprises the nucleotide sequence set forth in any one of SEQ ID nos. 15 to 28.

In a sixth aspect, the present invention relates to a vector comprising the sgRNA described above. Preferably, the vector comprises a T7 promoter and a fragment DNA of sgrnascfold.

Preferably, the vector is a viral vector, such as a lentivirus, retrovirus, adenovirus, herpes simplex virus, and the like.

The seventh aspect of the invention relates to a targeting vector, which comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a homonymous specific recombination site recognition sequence, a PHB1 gene nucleotide sequence and a homonymous specific recombination site recognition sequence from 5 'to 3'.

Preferably, the nucleotide sequence of the PHB1 gene comprises exons 4 to 5 of the PHB1 gene.

Preferably, the 5' arm is shown in SEQ ID NO. 51.

Preferably, the 3' arm is shown in SEQ ID NO. 52.

Preferably, the homotropic specific recombination site recognition sequence is LoxP or Frt.

Preferably, the targeting vector further comprises a marker gene, more preferably, the marker gene is a gene encoding a negative selection marker, and even more preferably, the gene encoding the negative selection marker may be a gene encoding diphtheria toxin subunit A (DTA), herpes simplex virus thymidine kinase (HSV-tk), SacB, rpsl (strA), tetAR, pheS, thy, CaCY, gata-I, ccdB, or the like.

In one embodiment of the present invention, the targeting vector further comprises a resistance gene selected from a positive clone, and more preferably, the resistance gene selected from the positive clone can be neomycin phosphotransferase coding sequence Neo, hygromycin B phosphotransferase (hph), xanthine/guanine phosphotransferase (gpt), hypoxanthine phosphotransferase (Hprt), thymidine kinase (tk), and puromycin acetyltransferase (puro).

An eighth aspect of the present invention relates to a method for preparing a vector for sgRNA, including the steps of:

1) preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence from any sgRNA target sequence with a sequence shown in SEQ ID NO. 1-14;

2) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, sequentially carrying out enzyme digestion on the fragments to be connected to a framework vector, and carrying out sequencing verification to obtain a pT7-sgRNA vector;

3) respectively synthesizing the forward oligonucleotide and the reverse oligonucleotide in the step 1), and denaturing and annealing the synthesized sgRNA oligonucleotides to form a double strand which can be connected into the pT7-sgRNA vector in the step 2);

4) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step 3) with pT7-sgRNA vectors, and screening to obtain sgRNA vectors.

In a ninth aspect, the present invention relates to a DNA molecule encoding the sgRNA described above.

A tenth aspect of the present invention relates to a cell containing the targeting vector, the sgRNA targeting PHB1 gene, the vector containing the sgRNA, or the DNA molecule.

An eleventh aspect of the present invention relates to a sgRNA that includes the targeting vector, the targeted PHB1 gene, the vector including the sgRNA, the DNA molecule, or the cell, and uses thereof for PHB1 gene knock-out.

In a twelfth aspect, the invention relates to an animal disease model, wherein the disease model is derived from the non-human animal or its offspring, and the non-human animal obtained by the above construction method or its offspring. Preferably, the disease comprises obesity, diabetes, choroid plexus related diseases, tumors or inflammation. Such as tumor-bearing animal models.

In a thirteenth aspect of the present invention, there is provided a method for preparing a disease model of an animal, said method comprising the steps of constructing the above non-human animal; preferably, the disease comprises obesity, diabetes, choroid plexus related diseases, tumors or inflammation, and further preferably, the step of implanting tumor cells is included.

In a fourteenth aspect of the present invention, there is provided a use of the sgRNA, the vector comprising the sgRNA, the DNA molecule, the targeting vector, the non-human animal or its progeny, or the non-human animal or its progeny obtained by the above-described construction method, for preparing a disease model of an animal, preferably, the disease includes obesity, diabetes, choroid plexus-related diseases, tumors, or inflammation.

In a fifteenth aspect of the present invention, the sgRNA, the vector comprising the sgRNA, the DNA molecule, the targeting vector, the non-human animal or its progeny, the non-human animal obtained by the above construction method or its progeny, or the disease model described above are provided for use in studying the toxicological effects of heavy metals or other toxic substances on the choroid plexus of the brain and in preparing a medicament for treating obesity, diabetes, choroid plexus-related diseases, tumors, and/or inflammation.

In a sixteenth aspect, the present invention relates to a cell, tissue or organ derived from the above-mentioned non-human animal or progeny thereof, or a disease model.

The seventeenth aspect of the present invention relates to an application of the above non-human animal or its progeny, the non-human animal obtained by the above construction method or its progeny, and the above cell, tissue or organ in the preparation of a medicament for treating or preventing obesity, diabetes, choroid plexus-related diseases, tumors or inflammation.

The eighteenth aspect of the present invention relates to an application of the above non-human animal or its progeny, the non-human animal obtained by the above construction method or its progeny, and the above cell, tissue or organ in research related to PHB1 gene or protein, wherein the application comprises:

A) to applications in the development of products involving cell cycle regulation;

B) as model systems for toxicology, pharmacology, immunology, microbiology and medical research;

C) relates to the production of cell cycle regulatory processes and the use of animal experimental disease models for the application in the aetiology, toxicology studies, for the development of diagnostic strategies or for the development of therapeutic strategies;

D) screening, drug effect detection, curative effect evaluation, verification or evaluation of PHB1 signal channel regulator in vivo; or,

E) research on PHB1 gene or protein function, research on medicine and drug effect aiming at PHB1 target site, and research on application of medicines for treating obesity, diabetes, choroid plexus related diseases, and tumor or inflammation.

The "choroid plexus-related disorders" described herein include, but are not limited to, choroidal cysts, choroidal enlargement, choroidal papillomas, meningiomas, ventriculitis, plexitis, Sturge-Weber syndrome, or neurofibromatosis.

The "tumor" according to the present invention includes, but is not limited to, lymphoma, brain cancer, non-small cell lung cancer, cervical cancer, esophageal cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

The term "inflammation" as used herein includes acute inflammation as well as chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

The term "treating" (or "treatment") as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treatment" or the like refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

The term "homology" as used herein refers to the fact that, in the aspect of using an amino acid sequence or a nucleotide sequence, a person skilled in the art can adjust the sequence according to the actual working requirement, so that the used sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

One skilled in the art can determine and compare sequence elements or degrees of identity.

The Cre-ER disclosed by the invention is a fusion protein (Cre-ER) positioned in cytoplasm and formed by fusing a ligand binding region (LBD) of a human estrogen receptor (ER for short) with a Cre recombinase, wherein the fused Cre protein can be subjected to conformational change and dissociated from an anchoring protein HSP90 only after hormone induction, enters a cell nucleus, recognizes a loxP site and is subjected to cutting.

The Cre-ERT provided by the invention is obtained by performing point mutation (G521R) in a ligand binding region of ER, and only responding to exogenous artificially synthesized estrogen, such as Tamoxifen (Tamoxifen), to induce Cre-ERT.

The Cre-ERT2 of the invention is that on the basis of Cre-ERT, 3 point mutations are added in LBD of ER: C400V/M453A/L544A, and the obtained mutant is Cre-ERT 2. Cre-ERT2 is more sensitive to tamoxifen metabolite 4-OHT.

The invention relates to an iCre-ERT2, which is based on Cre-ERT2, further improves Cre recombinase and aims to improve expression in mammals and reduce the chance of epigenetic silencing, and concretely relates to Shimshek DR, et al Codon-improved Cre restriction enzyme (iCre) expression in the gene, Gene 2002.

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal is selected from the group consisting of a mouse, a rat, and a hamster. In one embodiment, the non-human animal is selected from the murine family. In one embodiment, the genetically modified animal is from the family of cricotidae (e.g., mouse-like hamsters), cricotidae (e.g., hamsters, new world rats and mice, voles), muridae (true mice and rats, gerbils, spiny mice, crow rats), marmoraceae (mountaineers, rock mice, tailed rats, madagaska rats and mice), spiny muridae (e.g., spiny mice), and spale (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the non-human animal is selected from a mouse and a rat. In one embodiment, the non-human animal is a rat.

In a particular embodiment, the non-human animal is a non-human mammal which is a mouse strain selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6 6357 BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10 and C57BL/Ola, C57 36 BL, C58, Br, A/Ca, CBA/J, CBA/CBA, CBCBH/J, CBA, CBH.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mullisetal U.S. Pat. No.4, 683, 195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds., In-chief, Academic Press, Inc., New York), specific, volumes, 154 and 155 (Wuetal. eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Caloseds, 1987, Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The term Wildtype allel (WT) as described in this invention: i.e. wild type rats;

the term Targeted allel (fl) as described herein: i.e., F1 generation positive heterozygous rats obtained by mating F0 generation mice and wild type rats.

The term Deleted allele (Δ) as described herein: that is, rats with the objective gene knocked out in a tissue-specific or whole body manner are obtained by mating fl rats with tissue-specific Cre or Cre-deleter rats.

The invention has the technical effects that the CRISPR/Cas9 and Cre/loxP two-major gene recombination system is applied to gene conditional knockout recombination, and the new view point of choroid plexus tissue is taken as a research target: choroid plexus tissues play an important role in brain development, maturation, aging processes, endocrine regulation, and pathogenesis of some neurodegenerative diseases. In the toxic action and pharmacological action of exogenous substances, the choroid plexus has double characteristics, namely the nerve protection effect (preventing toxicant from entering brain tissues and cerebrospinal fluid); and some target tissues of neurotoxins (as sites of toxicant accumulation; or the toxic effects of exogenous substances can directly lead to structural and functional impairment of BCB). And based on a certain research basis, the gene of the functional protein PHB1 is directly modified, so that the action mechanism of PHB1 in the toxicity generation process can be better discussed, and the conditional knock of the gene of the brain choroid plexus PHB1 is not established unless a human animal model is established at present.

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

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

FIG. 1: technical roadmap for conditional knock-out rats for the PHB1 gene;

FIG. 2: southern blot detection of the F1 mouse tail DNA;

FIG. 3: the detection result of the activity of the sgRNA1-sgRNA14, wherein Con is a negative control, and PC is a positive control;

FIG. 4: schematic diagram of targeting strategy;

FIG. 5: schematic representation of Southern blot screening strategy;

FIG. 6: the enzyme digestion and sequencing verification result shows that 1 uses restriction enzyme KpnI, the length of the target fragment is 4116bp +2973bp, 2 uses restriction enzyme EcoRI, and the length of the target fragment is 5519bp +1138bp +432 bp; 3, using restriction enzyme as XhoI, the length of the target fragment is 4085bp +3004bp, and ck is a control group;

FIG. 7: genotype test results of F0 rat, wherein M is Marker, WT is wild control, H₂O is water control, A is (EGE-ZK-033) -L-GT-F/cKO-3'-DO-R primer detection result, B is cKO-5' -DO-F/(EGE-ZK-033) -R-GT-R primer detection result;

FIG. 8: f1 generation rat genotype test result, wherein M is Marker, WT is wild control, + is positive control, H₂O is water control, A is (EGE-ZK-033) -L-GT-F/cKO-3'-DO-R primer detection result, B is cKO-5' -DO-F/(EGE-ZK-033) -R-GT-R primer detection result;

FIG. 9: obtaining a tissue-specific PHB1 gene knockout rat scheme, wherein WT is a wild type rat, fl is an F1 generation positive heterozygote rat obtained by mating an F0 generation rat and a wild type rat, and delta is a tissue-specific or whole-body knockout rat obtained by mating an fl rat and a tissue-specific Cre or Cre-deleter rat;

FIG. 10: conditional PHB1 gene knockout first step protocol genotype mapping (one);

FIG. 11: conditional PHB1 gene knockout first step protocol genotype mapping (two);

FIG. 12: conditional PHB1 Gene knockout the second protocol genotype mapping;

FIG. 13: obtaining a PHB1 full gene knockout rat scheme, wherein WT is a wild type rat, fl is an F1 generation positive heterozygote rat obtained by mating an F0 generation rat and a wild type rat, and delta is a tissue specificity or whole body knockout rat obtained by mating an fl rat and a tissue specificity Cre or Cre-deleter rat;

FIG. 14: PHB1 full knockout first step protocol genotype mapping;

FIG. 15: the second step of the whole gene knockout of PHB1 is a genotype map;

FIG. 16: schematic diagram of pCS-3G vector;

FIG. 17: RNA electrophoresis at 65 deg.C for 5 min.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 conditional PHB1 Gene knockout Flox rats

In order to knock out a PHB1 gene, a rat PHB1 gene is knocked out mainly through a Cre/loxP recombination system, LoxP is inserted into a rat PHB1 locus through a CRISPR/Cas9 system to obtain a conditional PHB1 gene knock-out Flox rat, a recombinase or a recombinase expression sequence is introduced into the conditional PHB1 gene knock-out Flox rat, a conditional PHB1 gene knock-out Flox animal PHB1 gene is deleted, so that the PHB1 protein in choroid plexus tissues is not expressed or the expressed PHB1 protein does not have functions, and the PHB1 gene knock-out rat is obtained, and the specific construction method is shown in figure 1.

Firstly, a CRISPR/Cas system is introduced for gene editing, and target sequences in the system determine the target specificity of sgRNAs and the efficiency of inducing Cas9 to cut target genes, but the gene sequences may be different in different strains. In order to ensure the efficiency of the designed CRISPR/sgRNA, the target site sequence of the tail of a rat needs to be subjected to PCR amplification and sequencing verification to ensure that the recognition sequence of the sgRNA is completely consistent with the DNA sequence of the rat of the constructed strain. The PCR primers are shown in Table 1.

Efficient and specific target sequence selection and design are the premise of constructing an sgRNA expression vector, and a sgRNA target site sequence (sgRNA 1-sgRNA 14) is designed and synthesized, wherein the target site sequence targeted by each sgRNA is shown in Table 2, and the bottom strand oligonucleotide sequence of each sgRNA is shown in Table 3. Oligos were synthesized according to the designed sgRNA sequence and ligated into the pCS-3G plasmid by Gibson seamless ligation as shown in FIG. 16, and the ligation products were transformed and then submitted to sequencing for correct sequencing.

Table 1: PCR primer name and specific sequence

Table 2 sgRNA sequence listing

Table 3: bottom strand oligonucleotide sequence of sgRNA

Detection kit (UCA) independently developed by using Baiosai chart^TM) The sgRNA/Cas9 is subjected to activity detection, the activity detection result is shown in figure 3, and sgRNA5 and sgRNA8 are selected comprehensively from the sgRNA/Cas9 for subsequent experiments.

Next, CRISPR/Cas9 was transcribed in vitro, with the following specific steps:

1) preparing and obtaining a sgRNA forward oligonucleotide sequence and a reverse oligonucleotide sequence, wherein the forward oligonucleotide sequence and the reverse oligonucleotide sequence of sgRNA5 and sgRNA8 are respectively shown as SEQ ID NO.33-36, and the sequences are shown in Table 4;

2) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, sequentially carrying out enzyme digestion on the fragments to be connected to a skeleton vector, and carrying out sequencing verification to obtain a plasmid vector (pT 7-sgRNA) with a T7 promoter;

3) the forward oligonucleotide and the reverse oligonucleotide described in step 1) were synthesized separately, and the synthesized sgRNA oligonucleotides were denatured and annealed to form a double strand (SEQ ID NO: 15-28);

4) linking the double-stranded sgRNA oligonucleotides annealed in the step 3) with pT7-sgRNA vectors respectively, screening to obtain sgRNA vectors, and carrying out in vitro transcription to obtain RNA for microinjection, wherein the result is shown in FIG. 17.

Table 4: sgRNA5 and sgRNA8 forward and reverse oligonucleotide sequences

Next, sgRNA5 and sgRNA8 are selected to design and construct a PHB1 gene conditional knockout targeting vector by selecting sgRNA/Cas9 target site sequence information, and a targeting strategy as shown in fig. 4 is designed, wherein the targeting vector contains a rat PHB1 gene homologous sequence, 2 loxp sequences, a3 'arm and a 5' arm, and sequentially comprises a5 'arm (SEQ ID No. 51), loxp, a PHB1 gene homologous sequence (E4, 5), loxp and a 3' arm (SEQ ID No. 52). Wherein, the loxP sites at the 5 'end and the 3' end are respectively inserted into intron3 and intron 5; the homology arms of the 5 'end and the 3' end are 1423bp and 1437bp respectively.

The construction of the targeting vector can be carried out by a conventional method, such as enzyme digestion connection, direct synthesis and the like. The constructed targeting vector is verified by enzyme digestion identification and sequencing, and the results are shown in FIG. 6, wherein 1-3 are positive clones. The correct targeting vector will be verified for subsequent experiments.

Mixing an in-vitro transcription product of a sgRNA vector, Cas9 mRNA and a targeting vector to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of rat fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, detecting and confirming the introduction condition of the targeting vector by utilizing a PCR technology, and screening correct positive clone cells, wherein a PCR integration detection primer is shown in Table 5:

table 5: loxp sequence insertion detection primer

The selected correct positive clone cells are transplanted into the oviduct of a receptor mother mouse to develop, and F0 generation rats, namely Flox rats, are obtained. The genotype of the F0 rat is detected, the used PCR primers are shown in Table 6, the result is shown in FIG. 7, and EK33-30, EK33-36, EK33-39, EK33-46, EK33-52 and EK33-54 are positive mice of F0 generation by PCR amplification and product sequencing. The F1 generation of rats was obtained by mating F0 generation positive rats with wild type rats, and the F1 generation rats were subjected to PCR identification and sequencing detection, wherein the PCR detection primers are shown in Table 6. As shown in FIG. 8, 1EK33-21, 1EK33-28, 1EK33-38, and 1EK33-41 were confirmed by PCR and sequencing to be positive conditioned knockout rats of F1 generation.

Table 6: f0 and F1 generation rat detection PCR primer name and specific sequence

PCR reaction conditions (Touchdown):

the Southern blot assay was performed on rats identified as positive by F1 PCR to confirm the presence of random insertions.

A schematic of the Southern blot screening strategy is shown in FIG. 5.

The specific design is as follows:

introducing Southern blot enzyme cutting sites outside the loxP sites at the 5 'end and the 3' end respectively, and if correct recombination occurs, two bands of a wild type and a mutant type can appear; if not recombined correctly, only wild-type bands will appear.

TABLE 7 length of the particular probes and target fragments

TABLE 8 Probe Synthesis primers

The lengths of the specific probes and the target fragments are shown in tables 7 and 8, the detection results are shown in FIG. 2, and 1EK33-21, 1EK33-28, 1EK33-38 and 1EK33-41 are positive rats of the F1 generation, and all have no random insertion.

Example 2 conditional PHB1 Gene knockout rats

In order to obtain a rat expressing Cre recombinase in choroid plexus tissues, specifically, a nucleotide sequence which is regulated by tamoxifen and encodes iCre-ERT2 is knocked into the downstream of a rat Adgra3 gene promoter by using a CRISPR/Cas9 system, and the Cre recombinase expression in a rat body is regulated by an Adgra3 gene regulation element, so that the rat specifically expresses Cre recombinase in the choroid plexus tissues.

The Flox heterozygote rat obtained in example 1 is crossed with a rat expressing Cre recombinase in choroid plexus tissue to realize the tissue-specific knockout of the target gene, and the specific scheme is shown in FIG. 9. WT is wild type rat, fl is F1 generation positive heterozygote rat obtained by mating F0 generation rat and wild type rat, and delta is tissue-specific or whole body knockout rat obtained by mating fl rat and tissue-specific Cre or Cre-deleter rat.

The first step is as follows: mating with tissue-specific Cre rats (Ts-Cre) to obtain fl heterozygous rats, as shown in fig. 10 or 11; wherein the genotype detection strategy is shown in table 9:

TABLE 9 conditional knockout rat first-step genotype detection strategy

Only heterozygote rats (fl/+, Cre/+) can be obtained in this step, and homozygote rats (experimental rats) need to be further mated for obtaining; rats with genotypes +/+, Cre/+, fl/+, +/+ can be used as controls.

The second step is that: obtaining fl homozygote rats, and crossing the heterozygote rats (fl/+, Cre/+) obtained in the previous step with each other to obtain homozygote rats (fl/fl, Cre/+) as shown in fig. 12, wherein the genotype testing strategy is shown in table 10:

TABLE 10 conditional knockout rat second step genotype detection strategy

Rats with the genotype fl/fl and Cre/+ belong to the experimental group (i.e., conditional knockout rats), and rats with other genotypes belong to the control group, preferably the control group is +/+, Cre/+ rats.

The primers used in this example are shown in Table 5.

Example 3PHB1 Whole Gene knockout rat (Whole body PHB1 knockout rat)

To obtain full knockout rats, fl heterozygous rats were mated with Cre-deleter rats, and the mating schedule is shown in FIG. 13. WT is wild type rat, fl is F1 generation positive heterozygote rat obtained by mating F0 generation rat and wild type rat, and delta is tissue-specific or whole body knockout rat obtained by mating fl rat and tissue-specific Cre or Cre-deleter rat.

The first step is as follows: mating with a Cre-deleter rat to obtain a full gene knockout heterozygote rat; the resulting F1 generation heterozygote rats were mated with Cre-deleter rats to remove Exon 4-5. The Cre-deleter rats used may be homozygotes or heterozygotes, as shown in FIG. 14. The genotype detection strategy is shown in table 11:

TABLE 11 first-step genotype detection strategy for full-knockout rats

Only heterozygote rats (delta/+, Cre/+) can be obtained in the step, and homozygote rats for experiments need to be further mated and obtained; rats with a genotype of +/+, Cre/+ can be used as controls.

The second step is that: whole gene knockout homozygous rats were obtained and heterozygous rats (Δ/+, Cre/+) were further mated as shown in FIG. 15. Wherein the genotype detection strategy is shown in table 12:

TABLE 12 second-step genotype detection strategy for full-knockout rats

Rats with a genotype of Δ/Δ belong to the experimental group (i.e., whole knockout rats), and rats with genotypes of Δ/+ and +/+ are the control group.

The primers used in this example are shown in Table 5.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

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tgtcttttgc tgctgtggtt aatccttccc atcagttcct ggtccccaga gtgactgtcc 180

aaactagtct ttggcagttt gtctctcctt gtggctgaca actttccctt tgccttgttt 240

tccacggtct ctgctccagc agggcagacc tcagactgcc ctctcacagg ctgcgccaca 300

cagccctgtg cctgagatat gccactggat ttctagttgg agcctgaagt gaccagccct 360

taccattccc ctccccagtc agtgaaggca gtaaagggag ctgactcgtg ggaagaagag 420

ggatcccgtg gggctcacgg cactcgatgc cctcagagct gttcctgggc agatgtttgc 480

tccttgtgct gtgcaggctt gctgccaggt tgaaggctcc tgtacaggga tgaccaaaaa 540

gagagctaac tattaaggac ctcagtcctc aggagggcaa gcatgagaac cggtaataga 600

gcaaggagaa gatggccagg ggcaagtgga gcagaggaga tctgcctgct ctccctagtg 660

ctgacctctt gattggagaa cctagagtga gaagtcaggt cagagaagag aagagaagag 720

gggaatagga agtctgcctg ccgctcctgc agaatgctgg gcctggcttg cctgctgcca 780

agaggaaata gaaggtgggg tggggtgatg gggatgagtg aggttggatt tagaatcctg 840

cttctaaacg tccgcctttg cgctttatat ccgtgttgtc tcatggctgt gctaagtcgt 900

atttgtacct gcacatgcta aaaattggga cgggcgattc tagtcatgaa tttcccactt 960

ctctcagtgg ctgggtcagg actccaaccc ctggcagccc ttagctgctg gtgctggcga 1020

acttacacag ggtggctgtg ccttagccct gcacgggcct ggattgtcag gagtaggtag 1080

gggctgactg tctccccaga tgcttgtgga caatgagtct tttgaattcg ccctgccttt 1140

ggcttaacgg cctttgtttt tagactttat cagaaatcct ctcacatcag cgtgctgtag 1200

tgccagctgc caccttctct caggtatcag gaagcaggcc ctggatggat gagccttagt 1260

ctactatcta tgagaaggtg ctgtccccat ttctcctcat ctcacagact aatacactca 1320

gtttgactaa gactggtccc caccattaga aaacagcaca gtggtaccac agcccaggtc 1380

agtggactcc atggatggac gaagtttcac cagatccaag taa 1423

<210> 52

<211> 1437

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

ggaaggcagt ataggaaata gagcatttca caccaaggcg aggggaaggg gtcgtgtttc 60

tagagtctct gggaaaagtc ttggagtgat ttgtcgggta acaaggcaga gtgcttcctg 120

ttctctctgc ggcctgcctg ggaaaggcta ggcttcccct cagcatctgc acagctgttc 180

aaacagccat tctctgagca acctctgctc ccctccaggc agggcctctt tcctccctcc 240

ctctctccgg ggaagggcat gtggctccct ctcctggcac tatgccaggc tgcagtcctt 300

gactctgcag cctgccttgc tccaggctct cccatgttaa cacagcactc tggcagtgag 360

aacacacgga gtaaaaacct gagagaaact tggcatcttt ctagactctg accacatact 420

ctatcctggg gaaatgtggc ctgaccacaa gagaccatag ctaagaggtg tgaggagaaa 480

attagatttc ctggggaatt tgctgccctc agctggccct ttcagaaaga aattttattc 540

tttaattaca catttagctt ttcagttctc aggattgtgt ttccaaatcc aaacttcaaa 600

tgagagtcca gagtgggtcc gacagagtag agcagcggca agataagatt tagctggcgt 660

atttggcaca cgcctggaat ggccgtgcgt atcagccctc ctgggctcag agtaagaccg 720

tcttagagca gaaacaagcc aaaagactta ggtctgtatt gtggaaaact cgggaggatg 780

tgtggtcaag atttttgcct gtacaaagca gagtttggga gggagcaggg gatgggggat 840

gaatgaagca aaattgcatt tgaacctaaa ttaacacctt tagaattttt tcctagactt 900

atattttttt tagctcgcat gtatgttttg tccgtgtttg taagggtgcc acgtgtgtgc 960

atcagatacc ctgtaactag agttagagat ggtttgaatc accatgtggc tgctaagcag 1020

caaacctggc cctttgcaag agcagccagg gctcctaagc actgagccct gtgcctgctc 1080

ttgcaataga aagatggact cagtgacttc ctgtaggctg ggccttagga gtgccaaggt 1140

ctgtccctct tctgtgctac ctcagagatc gtccctccag ccaaatactc gagctattta 1200

tgtgataggg aaggtctgtt gacagtcagg ctctcctgag caatggcttc acctgccttt 1260

cttttaaaac ctttaccaga actagtgtgt gtcagggatg ccatgatctg tgggccaccc 1320

tctgtcttcc tgtcaagggt ggaggtgctc tgagaaggaa gtgggctggt taatccataa 1380

gcagagaccg ccaggctcct ccatccctgg gaaccagctg catcccttgc tctcagg 1437

Claims (8)

1. A construction method of PHB1 gene knock-out non-human animal is characterized in that the construction method comprises knocking out No.4 to No.5 exons of PHB1 gene, so that PHB1 protein in choroid plexus tissue of the non-human animal is not expressed or the expressed PHB1 protein does not have function;

the construction method comprises the steps of inserting a homodromous specific recombination site recognition sequence into a No.3 intron and a No.5 intron of a non-human animal PHB1 gene respectively by using sgRNA to obtain a conditional PHB1 gene knockout Flox non-human animal, and introducing a recombinase or a recombinase expression sequence into the conditional PHB1 gene knockout Flox non-human animal; the target site sequence of the 5 'end of the sgRNA targeting is shown in SEQ ID NO.5, and the target site sequence of the 3' end of the sgRNA targeting is shown in SEQ ID NO. 8;

the non-human animal is a rat or a mouse.

2. The construction method of claim 1, wherein the construction method comprises inserting a LoxP or Frt sequence into intron3 and intron5 of PHB1 gene of non-human animal to obtain conditional PHB1 gene knockout Flox non-human animal, mating conditional PHB1 gene knockout Flox non-human animal with non-human animal expressing Cre or Flp recombinase in choroid plexus tissue, and knocking out PHB1 gene of non-human animal by Cre-LoxP or Flp-Frt gene recombination system.

3. The method according to claim 2, wherein the non-human animal expressing Cre or Flp recombinase in choroid plexus tissue is obtained by knocking a nucleotide sequence encoding Cre into a gene encoding a protein expressed from choroid plexus tissue.

4. The method of claim 1, comprising inserting a LoxP or Frt sequence into the PHB1 locus using sgRNA and/or targeting vectors,

the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a homodromous specific recombination site recognition sequence, a PHB1 gene nucleotide sequence and a homodromous specific recombination site recognition sequence from 5 'to 3'; wherein, the PHB1 gene nucleotide sequence comprises exons 4 to 5 of PHB1 gene, the 5 'arm is shown as SEQ ID NO.51, and the 3' arm is shown as SEQ ID NO. 52; the homotropic specific recombination site recognition sequence is LoxP or Frt.

5. A construction method of a conditional Flox non-human animal knocked out by a PHB1 gene is characterized in that a specific recombination site recognition sequence in the same direction is inserted into an intron3 and an intron5 of a non-human animal PHB1 gene by using sgRNA, the conditional Flox non-human animal knocked out by the PHB1 gene is obtained, a target site sequence at the 5 'end of the sgRNA targeting is shown as SEQ ID No.5, a target site sequence at the 3' end of the sgRNA targeting is shown as SEQ ID No.8, and the non-human animal is a rat or a mouse.

6. The sgRNA of a targeted non-human animal PHB1 gene is characterized in that a target site sequence at the 5 'end of the sgRNA is shown as SEQ ID No.5, a target site sequence at the 3' end of the sgRNA is shown as SEQ ID No.8, and the non-human animal is a rat or a mouse.

7. A targeting vector, characterized in that the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a specific recombination site recognition sequence in the same direction, a PHB1 gene nucleotide sequence and a specific recombination site recognition sequence in the same direction from 5 'to 3'; the nucleotide sequence of the PHB1 gene comprises exons 4 to 5 of the PHB1 gene, the 5 'arm is shown as SEQ ID NO.51, the 3' arm is shown as SEQ ID NO.52, and the homodromous specific recombination site recognition sequence is LoxP or Frt.

8. Use of the non-human animal obtained by the construction method according to any one of claims 1 to 5 or its progeny, the sgRNA according to claim 6, or the targeting vector according to claim 7 for studying the toxicological effects of heavy metals or other toxic substances on the choroid plexus of the brain and for preparing a medicament for treating or preventing obesity, diabetes, choroid plexus-related diseases, tumors, or inflammation.

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