CN114107401A - Construction method and application of transgenic non-human animal - Google Patents

Abstract

The invention relates to a construction method of a transgenic non-human animal and application thereof in the field of biomedicine, wherein a non-human animal model of an intestinal tract specificity expression NPC1L1-FLAG-EGFP fusion protein is constructed by adopting a CRISPR/Cas9 system, NPC1L1 can be traced in vivo, an action mechanism of NPC1L1 in cholesterol absorption is explored in vivo, and the construction method has important application value for research and development of medicaments for treating cholesterol-related diseases.

Description

Construction method and application of transgenic non-human animal

Technical Field

The application relates to a construction method and application of a transgenic non-human animal, in particular to a construction method based on a transgenic non-human animal and application thereof in the field of biomedicine.

Background

Cholesterol is an important structural component of mammalian cell membranes, playing an important role in cell signal transduction, intracellular transport, synthesis of steroid hormones and bile acids. Atherosclerotic cardiovascular disease is a major lethal disease in developed countries, and clinical and animal studies indicate that high levels of plasma cholesterol are an independent risk factor for atherosclerotic cardiovascular disease. Therefore, how to regulate cholesterol metabolism has attracted increasing researchers' attention.

Excessive intake of dietary cholesterol is a common cause of hypercholesterolemia, and understanding of the cholesterol absorption mechanism is expected to provide a new idea for the prevention and treatment of hypercholesterolemia. It is currently believed that extracellular free cholesterol absorption is primarily achieved by the vesicular endocytosis (vesicular endocytosis) pathway mediated by Niemann-Pick C1-Like protein 1(Niemann-Pick C1-Like 1, NPC1L 1). The NPC1L1 protein is an N-glycosylated protein consisting of 1333 amino acids and 13 transmembrane segments. It has a typical N-terminal signal peptide that selectively binds cholesterol and oxysterol, and a less conserved C-terminal cytoplasmic tail with an endocytic signal sequence essential for cholesterol internalization. The expression pattern of this protein is species and tissue specific.

NPC1L1 is highly expressed in the brush border of small intestine absorptive epithelial cells and the biliary tract of human and primate hepatocytes. The absorption rate of NPC1L1 knockout mice to cholesterol is reduced by about 70 percent, and the mice can resist diet-induced hypercholesterolemia; while the absorption of other lipids (e.g. triglycerides, phospholipids) and phytosterols is not affected. The analysis of cell molecular mechanisms of NPC1L1 vesicle formation, endocytosis, transportation, circulation and other related processes has important significance for disclosing cholesterol absorption mechanisms.

The search for a co-acting molecule of NPC1L1 is an effective strategy for analyzing the cholesterol absorption mechanism. Since rodent liver tissues including rats hardly express NPC1L1 protein, studies on the mechanism of NPC1L 1-mediated cholesterol absorption have been conducted by introducing human NPC1L1 gene into isolated rat hepatoma cells as a model.

Since liver tissues of rodents including mice and rats rarely express NPC1L1 protein, in vitro studies of the mechanism of NPC1L1 in cholesterol absorption were mainly performed by overexpressing human NPC1L1 protein in rat liver cancer cell lines. Although the exogenous NPC1L1 gene can simulate the function of the protein to a certain extent, compared with a cell model expressing the endogenous NPC1L1 protein, the expression level is uncontrollable, so that the method has obvious limitation on the aspect of clarifying a signal path.

Non-patent literature "Flotillins plant an addressing role in Niemann-Pick C1-like 1-mediated cholestol uptake" utilizes a cell model of NPC1L1-EGFP over-expression in rat hepatoma cells, and finds that the co-acting molecule Flotilin plays an important role in mediating and forming a membrane structure of an NPC1L1 micro-region (microdomain) rich in cholesterol and absorbing the cholesterol through immunoprecipitation and mass spectrometry. However, there is no animal model for in vivo tracking of NPC1L1 to further explore the mechanism of action of NPC1L1 in cholesterol absorption in vivo.

In the aspect of in vivo function research of the NPC1L1 protein, a currently common mouse model is an NPC1L1 gene whole-body knockout model, or a villin-Cre is utilized to enable the intestinal tract to overexpress the human NPC1L1 or an apolipoprotein E promoter is utilized to overexpress the human NPC1L1 in the liver on the basis of whole-body knockout, so as to investigate the effect of the intestinal tract or liver NPC1L1 protein in cholesterol metabolism. Although the location of the knocked-in exogenous NPC1L1 in the target tissue is basically consistent with that of the endogenous protein, the phenomenon of off-target can also occur. The studies reported in the prior literature on the localization of NPC1L1, both in vivo mouse tissue and in vitro cell models, were visualized by fluorescently labeled secondary antibody hybridization, which amplified the fluorescent signal and also generated a mixed signal. Patent document CN101580871A discloses the use of reporter gene and the ligation of tag protein for the localization of NPC1L1 protein, but its insertion of tag protein between amino acids encoding NPC1L1 protein may affect the expression and function of NPC1L1 protein, and this patent document also uses in vitro tracing method.

Disclosure of Invention

According to the application, a CRISPR/Cas9 genome editing technology is utilized to construct a mouse model for specifically expressing NPC1L1-FLAG-EGFP fusion protein in an intestinal tract, and enhanced green fluorescent protein EGFP and FLAG protein labels are respectively marked at the C end of endogenous NPC1L1 protein.

In a first aspect, the present invention relates to a method for constructing a transgenic non-human animal, which comprises inserting a reporter gene and/or a gene encoding a tag protein into the 3' non-coding region of the NPC1L1 gene of the transgenic non-human animal.

Preferably, the reporter gene includes, but is not limited to, chloramphenicol acetyl transferase gene (CAT), human growth hormone gene (hGH), secreted alkaline phosphatase gene (SEAP), green fluorescent protein Gene (GFP), enhanced green fluorescent protein gene (EGFP), beta-galactosidase gene (beta-Gal), TdTomato red fluorescent protein gene (TdTomato: tomato tandem dimer), mCherry red fluorescent protein gene (mCherry), or firefly luciferase gene. Further preferably, the reporter gene is EGFP, and the amino acid sequence of the EGFP protein is as shown in SEQ ID NO: shown at 31.

Preferably, the tag protein includes, but is not limited to, Myc tag protein, FLAG tag protein, His6 tag protein, T7 tag protein, V5 tag protein, HA tag protein or GST tag protein. Further preferably, the tag protein is a FLAG tag protein, and the amino acid sequence of the FLAG tag protein is as shown in SEQ ID NO: shown at 21.

Preferably, the non-human animal expresses a fusion protein of NPC1L1 protein-tag protein-reporter gene encoding protein. The NPC1L1 protein and the tag protein, and the tag protein and the reporter gene coding protein can be directly connected or connected through a linker. The linker may be a flexible linker, a rigid linker, a cleavable linker or a nonsense amino acid, etc., which are conventional in the art. Preferably a linker peptide. Preferably, the connecting peptide sequence is as shown in SEQ ID NO: shown at 30.

Further preferably, the non-human animal expresses a fusion protein of NPC1L1 protein-linker peptide-tag protein-linker peptide-reporter gene encoding protein.

In a specific embodiment of the invention, the non-human animal expresses NPC1L1-FLAG-EGFP (NPC1L1-FG) protein or NPC1L 1-linker peptide-FLAG-linker peptide-EGFP protein.

Preferably, the fusion protein is an intestinal tract specific expression protein. Further preferably, the intestine is the small intestine, such as the duodenum, jejunum or ileum.

Preferably, the insertion site is before the 3' non-coding region (preferably immediately before the 3' non-coding region), after the 3' non-coding region (preferably immediately after the 3' non-coding region), or between any two bases adjacent in the nucleotide sequence of the 3' non-coding region.

Further preferably, the insertion site is located between the end of the reading frame of the NPC1L1 protein and the 3' UTR. In one embodiment of the invention, the insertion site is located between the end of the reading frame of the NPC1L1 protein and the stop codon.

Preferably, the construction method includes constructing a transgenic non-human animal using the sgRNA and/or the targeting vector.

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

Further preferably, the sgRNA target site sequence is as set forth in SEQ ID NO: 13-20, preferably, the sgRNA sequence is as shown in SEQ ID NO: 5 to 12.

Still further preferably, the sgRNA sequence is as set forth in SEQ ID NO: 5, the sequence of the sgRNA target site is shown as SEQ ID NO: shown at 13.

Further preferably, the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, the donor DNA sequence comprising a nucleotide sequence encoding a tag protein and a reporter gene. Wherein, the nucleotide sequence coding the tag protein is directly connected with the reporter gene or connected with the reporter gene through the nucleotide sequence coding the linker. Preferably, the linker may be a flexible linker, a rigid linker, a cleavable linker, or a nonsense amino acid, etc., which are commonly used in the art.

In one embodiment of the invention, the linker is a connecting peptide.

In one embodiment of the present invention, the donor DNA sequence is a nucleotide sequence encoding a tag protein, a reporter gene, and a nucleotide sequence encoding a linker peptide (linker). More preferably, the donor DNA sequence comprises a cDNA sequence encoding a tag protein, encoding a linker peptide and a reporter gene.

Preferably, the donor DNA sequence comprises from 5 'to 3' FLAG-EGFP.

Preferably, the donor DNA sequence comprises a linker-FLAG-linker-EGFP from 5 'to 3'.

In one embodiment of the present invention, it is further preferred that the donor DNA sequence is as set forth in SEQ ID NO: as shown at 29.

Still more preferably, the 5' arm is as set forth in SEQ ID NO: 3, respectively.

Still more preferably, the 3' arm is as set forth in SEQ ID NO: shown at 28.

The non-human animal of the invention is selected from rodent, pig, rabbit, monkey and other non-human animals 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 introducing the targeting vector, sgRNA targeting NPC1L1 gene and Cas9 into non-human animal cells, culturing the cells (preferably embryonic stem cells), transplanting the cultured cells into oviducts of female non-human animals, allowing the cells to develop, and identifying and screening to obtain transgenic non-human animals.

In a second aspect, the present invention relates to a transgenic non-human animal or its offspring obtained by the above construction method.

The third aspect of the invention relates to sgRNA targeting an NPC1L1 gene, wherein the sequence of the target site of the sgRNA is shown in SEQ ID NO: any one of 13 to 20.

Preferably, the sgRNA sequence is as set forth in SEQ ID NO: 5 to 12.

In a specific embodiment of the present invention, the sgRNA sequence is as set forth in SEQ ID NO: 5, the sequence of the sgRNA target site is shown as SEQ ID NO: shown at 13.

In a fourth 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.

In a fifth aspect, the invention relates to a targeting vector comprising a5 'arm, a 3' arm and a donor DNA sequence. The donor DNA sequence includes the nucleotide sequence of coded label protein and reporter gene. Wherein, the nucleotide sequence coding the tag protein is directly connected with the reporter gene or connected with the reporter gene through the nucleotide sequence coding the linker. Preferably, the linker may be a flexible linker, a rigid linker, a cleavable linker, or a nonsense amino acid, etc., which are commonly used in the art.

In one embodiment of the invention, the linker is a connecting peptide. Preferably, the connecting peptide sequence is as shown in SEQ ID NO: shown at 30.

In one embodiment of the present invention, the donor DNA sequence is a nucleotide sequence encoding a tag protein, a reporter gene, and a nucleotide sequence encoding a linker peptide (linker).

More preferably, the donor DNA sequence comprises a cDNA sequence encoding a tag protein, encoding a linker peptide and a reporter gene.

Preferably, the donor DNA sequence comprises from 5 'to 3' FLAG-EGFP.

Preferably, the donor DNA sequence comprises a linker-FLAG-linker-EGFP from 5 'to 3'.

In one embodiment of the present invention, it is further preferred that the donor DNA sequence is as set forth in SEQ ID NO: as shown at 29.

Preferably, the reporter gene includes, but is not limited to, chloramphenicol acetyl transferase gene (CAT), human growth hormone gene (hGH), secreted alkaline phosphatase gene (SEAP), green fluorescent protein Gene (GFP), enhanced green fluorescent protein gene (EGFP), beta-galactosidase gene (beta-Gal), TdTomato red fluorescent protein gene (TdTomato: tomato tandem dimer), mCherry red fluorescent protein gene (mCherry), and firefly luciferase gene. Further preferably, the reporter gene is EGFP, and the amino acid sequence of the EGFP protein is as shown in SEQ ID NO: shown at 31.

Preferably, the tag protein includes, but is not limited to, Myc tag protein, FLAG tag protein, His6 tag protein, T7 tag protein, V5 tag protein, HA tag protein, GST tag protein, luciferase, or β -gal enzyme. Further preferably, the tag protein is a FLAG tag protein, and the amino acid sequence of the FLAG tag protein is as shown in SEQ ID NO: shown at 21.

Still more preferably, the 5' arm is as set forth in SEQ ID NO: 3, respectively.

Still more preferably, the 3' arm is as set forth in SEQ ID NO: shown at 28.

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).

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

The seventh aspect of the present invention relates to a cell containing the targeting vector, the sgRNA targeting the NPC1L1 gene, or the vector containing the sgRNA.

In an eighth aspect, the present invention relates to a sgRNA that includes the targeting vector, the targeting NPC1L1 gene, the vector including the sgRNA, or use of the cell for NPC1L1 gene modification.

In a ninth 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 construction method or its offspring. Preferably, the disease comprises cholesterol related diseases.

In a tenth aspect, the present invention relates to the use of the sgRNA described above, the vector comprising the sgRNA described above, the DNA molecule described above, the targeting vector described above, or the non-human animal described above or its progeny, for the preparation of a disease model for an animal, preferably, the disease comprises a cholesterol-related disease.

An eleventh aspect of the present invention relates to the sgRNA described above, the vector containing the sgRNA described above, the DNA molecule described above, the targeting vector described above, the non-human animal described above or its offspring, the non-human animal obtained by the above-mentioned construction method or its offspring, or the disease model described above, for use in vivo tracing of NPC1L1 protein and in the preparation of a medicament for treating or preventing cholesterol-related diseases.

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

In a thirteenth aspect, the invention relates to a fusion protein, which comprises a non-human animal NPC1L1 protein, a FLAG tag protein and an EGFP protein from N end to C end.

Preferably, the non-human animal NPC1L1 protein and the FLAG tag protein, the FLAG tag protein and the EGFP protein can be directly connected or connected through a linker. The linker may be a flexible linker, a rigid linker, a cleavable linker or a nonsense amino acid, etc., which are conventional in the art. Preferably a linker peptide. Preferably, the connecting peptide sequence is as shown in SEQ ID NO: shown at 30.

Further preferably, the fusion protein is NPC1L1-FLAG-EGFP protein or NPC1L 1-connecting peptide-FLAG-connecting peptide-EGFP.

In a fourteenth aspect of the present invention, there is provided a nucleic acid encoding the above-described fusion protein.

In a fifteenth aspect, the invention relates to a cell expressing the above fusion protein or comprising the above nucleic acid encoding the fusion protein.

Cholesterol-related disorders described herein include, but are not limited to, hyperlipidemia, atherosclerosis, coronary heart disease, hypertension, stroke, obesity, osteoporosis, senile dementia, cholelithiasis, hypocholesterolemia, or tumors.

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.

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

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 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); mulliserial.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, Vols.154and 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 technical effects of the invention are that a CRISPR/Cas9 gene recombination system is used for knocking in FLAG and EGFP genes, an NPC1L1-FLAG-EGFP non-human animal model is constructed, the NPC1L1-FLAG-EGFP protein is specifically expressed in the intestinal tract of the non-human animal model, the motion track of the NPC1L1 protein in the non-human animal body can be visually tracked under an EGFP label protein mirror, and the NPC1L1 protein and potential coaction proteins thereof are enriched and captured by using a FLAG label antibody. According to the technical scheme, the expression distribution of endogenous NPC1L1 protein cannot be changed, so that the experimental artifact caused by the expression of NPC1L1 outside target tissues can be effectively avoided. Meanwhile, the model can conveniently and directly observe the positioning of endogenous NPC1L1 protein, the formation of endocytic vesicles and the transportation of endocytic vesicles in cells by using a fluorescence microscope, and can also avoid the interference of a mixed signal caused by antibody hybridization. At present, no animal model capable of tracing NPC1L1 in vivo is available, so as to further explore the action mechanism of NPC1L1 in cholesterol absorption in vivo.

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: FIG. 1A is a UCA^TMDetecting the activity of sgRNA by using a CRISPR/Cas9 luciferase method, wherein fig. 1B shows that gel electrophoresis is used for detecting in vitro transcription products of sgRNA, wherein Con is a negative control, PC is a positive control, and M is Marker;

FIG. 2: FIG. 2A is a schematic diagram showing the construction of a targeting vector in which a FLAG-EGFP gene is inserted into the untranslated region on the 3' end of the NPC1L1 gene; 5' probe and EGFP probe show the binding position of the probe used for Southern blot detection; P1F/P1R, P2F/P2R and P3F/P3R show the positions of primers for identifying mouse genotypes; e18: exon 18; e19: exon 19; LA: a left homology arm; RA: a right homology arm; BspHI and BamHI are restriction enzymes; FIG. 2B: the enzyme digestion identification result of the correctly recombined targeting vector; ApaI, HindIII, DraIII, Eco47III and KpnI are restriction enzymes;

FIG. 3: FIG. 3A: southern blot detecting the gene homologous recombination condition of F1 mouse; mouse numbers are shown above each lane; 5' Probe; EGFP Probe: an EGFP probe; FIG. 3B: identifying the mouse genotype by a PCR method; knock-in allel: knock-in alleles; wild-type alloy: a wild-type allele; +/- + is a wild type mouse; T/T is NPC1L1-FLAG-EGFP homozygous mouse;

FIG. 4: localization of NPC1L1-FLAG-EGFP protein along the duodenum-ileum axis, HCD high cholesterol diet, scale: 50 μm;

FIG. 5: FIG. 5A is the tissue distribution of NPC1L1-FLAG-EGFP protein, and FIG. 5B is the distribution of NPC1L1-FLAG-EGFP protein in the small intestine;

FIG. 6: NPC1L1-FLAG-EGFP knock-in mice (T/T) showed similar phenotypes as wild type mice (+/+) in terms of body weight (a), food intake (B), blood glucose (C), plasma TC and TG (D & E), intestinal appearance (F), intestinal TC and TG (G & H), liver appearance and weight (I & J), liver TC and TG (K & L), wherein TC is total cholesterol, TG is triglyceride, HCD is high cholesterol diet, { p <0.05 }, { p < 0.01;

FIG. 7: visualization of cholesterol-induced endocytic vesicles in NPC1L1-FLAG-EGFP knock-in mice (T/T), wherein results were obtained from 5-30 minutes (a) with 200 μ L ink administered by gavage, 15 minutes (B) with 200 μ L corn oil containing varying concentrations of cholesterol, 0-60 minutes (C) with 200 μ L corn oil containing varying concentrations of cholesterol 20mg/ml cholesterol; (D) and (E): average number of NPC1L1-FLAG-EGFP containing vesicles under the jejunal brush border membrane, and a plurality of images (n-8-10) were taken and counted from each mouse (n-2-3), scale bar: 10 mu m;

FIG. 8: ezetimibe (Ezetimibe) inhibits the transport of cholesterol-containing endocytic vesicles, wherein a: fluorescence visualization results; b: average number of vesicle-containing NPC1L 1-FLAG-EGFP. A plurality of images (n 10) were taken and counted from each mouse (n 4), scale: 10 mu m;

FIG. 9: pCS-3G plasmid map.

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 construction of NPC1L1-FLAG-EGFP mice

In order to obtain NPC1L1-FLAG-EGFP transgenic mice, FLAG-EGFP coding genes are inserted into NPC1L1 genes through a CRISPR/Cas9 system, and transgenic mice expressing NPC1L1-FLAG-EGFP proteins are obtained.

Firstly, a CRISPR/Cas system is introduced for gene editing, a target sequence in the system determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut a target gene, according to the cutting principle of the sgRNA in the CRISPR/Cas9 system, a sgRNA sequence with high targeting fraction and low off-target rate is selected on a CRISPR design website (http:// www.sanger.ac.uk/htgt/wge /) aiming at an untranslated region behind a termination codon of an NPC1L1 gene, and a base mismatched with the target DNA sequence is introduced at the 3' end. sgRNA sequences and corresponding targeting sequences are shown in table 1. Homology arm sequences complementary to the pCS-3G backbone plasmid were added to both sides of the sgRNA sequence, with the left homology arm sequence being 5'-CTATTTCTAGCTCTAAAAC-3' (SEQ ID NO: 1) and the right homology arm sequence being 5'-GGTGTTTCGTCCTTTCCA-3' (SEQ ID NO: 2). Synthesizing single-chain oligonucleotide fragment, annealing, joining into pCS-3G plasmid linearized by XhoI and BamHI through Gibson seamless connection, finally obtaining pCS-3G plasmid map as shown in FIG. 9, selecting correct clone verified by sequencing after connection product conversion, adopting UCA^TMThe CRISPR/Cas9 luciferase activity detection method detects the activity of Cas9/sgRNA, the activity result is shown in figure 1A, and sgRNA1, sgRNA3, sgRNA5 and sgRNA8 all have strong shearing guide activity. And comprehensively considering the target position and activity result, selecting the sgRNA1 for in vitro transcription to prepare the guide RNA. Homologous arm sequences matched with a skeleton plasmid are respectively added on two sides of a DNA sequence corresponding to the sgRNA1, wherein the 5 'end sequence is 5'-CTATTTCTAGCTCTAAAAC-3'(SEQ ID NO: 1), and the 3' end sequence is 5'-TATAGTGAGTCGTATTA-3' (SEQ ID NO: 4). The two synthesized complementary single-stranded oligonucleotide fragments are annealed and assembled into plasmid vector pT7-2G which is cut by XhoI and BamHI in a Gibson seamless cloning mode, and after the plasmid is correctly sequenced, T7 RNA polymerase and MEGAshortscript are used^TMT7 transcription kit, in vitro transcription of sgRNA and Cas9 mRNA, product via MEGAclear^TMAnd after the transcription purification kit is purified and recovered, carrying out RNA electrophoresis detection. Gel electrophoresis showed that sgRNA1 appeared as a single band when incubated at 65 ℃ for 5min, suggesting successful preparation of RNA for microinjection (fig. 1B).

TABLE 1 sgRNA sequence targeting 3' UTR of NPC1L1 gene and targeting DNA sequence thereof

Next, the sequence information of the sgRNA/Cas9 target site of sgRNA1 was selected to design and construct targeting vectors, and the targeting strategy for knocking in the FLAG-EGFP coding sequence before the stop codon of the mouse NPC1L1 gene (between the end of the reading frame of the mouse NPC1L1 protein and the 3' UTR) is shown in FIG. 2A, in which the FLAG protein tag consists of 8 amino acids of DYKDDDDK (SEQ ID NO: 21), and the primers used for constructing the targeting vectors are shown in Table 2. Using genome DNA as template, using primer LA-F/RPCR to amplify 5' end homology arm (LA) (SEQ ID NO: 3), the size of product is 1388 bp; using genome DNA as a template, using an amplification product of a primer RA-F1/R as the template, and then amplifying by using a primer RA-F2/R to obtain a 3' end homology arm (RA) (SEQ ID NO: 28), wherein the size of the product is 1294 bp; the inserted FLAG-EGFP gene sequence (FG), the donor DNA sequence (SEQ ID NO: 29), was amplified by primers FG-F/R. The amplification product is subjected to AIO^TMCloning into a vector pTV-4G by the method, carrying out enzyme digestion and DNA sequencing for identification, wherein the enzyme digestion identification result of the targeting vector is shown in figure 2B, and 6076bp and 800bp fragments can be obtained by correct cloning through ApaI enzyme digestion; obtaining 3842bp, 2025bp and 1009bp fragments by HindIII and SphI double enzyme digestion; the 4611bp and 2265bp fragments can be obtained by double enzyme digestion of Eco47III and KpnI. The correct clones were used for subsequent microinjection.

sgRNA prepared by in vitro transcription, Cas9 mRNA and targeting vector plasmid are prepared into a mixture, the mixture is injected into the cytoplasm of a fertilized egg of a C57BL/6N mouse by a microinjection method and then is transplanted into a pseudopregnant recipient female mouse, and a son mouse obtained by the production of the female mouse is called an F0 generation mouse.

And (4) after the fertilized egg transplanted pseudopregnant female rat is raised in a single cage for 20 days, observing the birth condition of the suckling rat. After 1 week, the tissue of the tip of the F0 mouse tail was excised 1cm and dissolved in 500. mu.L of a genomic DNA lysate (0.2% SDS, 0.2mol/LNaCl, 5 mm)ol/L EDTA、0.1mol/LTris-HCl[pH 8.0]0.5mg/mL proteinase K), extracting genomic DNA, and carrying out PCR to identify the genotype. The positions of the design of the genotype identifying primers are shown by reference to P1F/P1R, P2F/P2R and P3F/P3R in FIG. 2A, and the reaction system comprises 1 muL (50ng) of genomic DNA, 2 XPCR mix12.5 muL, 1 muL of each of 10 mumol/L upstream and downstream primers, and ddH₂O9.5. mu.L, total volume 25. mu.L. Adopting Touchdown PCR, the reaction condition is 94 ℃, 2 min; the temperature is reduced by 0.7 ℃ in each cycle for 2min at 98 ℃, 10s, 67-57 ℃, 30s, 68 ℃ and 15 cycles; 25 cycles of 98 ℃, 10s, 57 ℃, 30s, 68 ℃ and 2 min; then at 68 ℃ for 10 min. After the reaction is finished, 10 microliter of PCR product is run for 2% agarose gel electrophoresis, and the mouse genotype is judged according to the size of the product and confirmed by sequencing. The 2154bp product was obtained by amplification with P1F/P1R primers and the 1971bp product was obtained by amplification with P2F/P2R primers and was a F0 positive mouse. P1F sequence is 5'-ACTTCTCTGTAGATGGAACCCAG-3' (SEQ ID NO: 22), P1R sequence is 5'-GTAGTTGTACTCCAGCTTGTGCCCC-3' (SEQ ID NO: 23); P2F sequence is 5'-CACGACTTCTTCAAGTCCGCCATGC-3' (SEQ ID NO: 24), P2R sequence is 5'-CAGCCATAAGACCCATGCCAG-3' (SEQ ID NO: 25); P3F sequence was 5'-CAGGGCCAGATGTTAACCAAGCTCT-3' (SEQ ID NO: 26) and P3R sequence was 5'-GTACCACTGCCACACGTTCCCAAG-3' (SEQ ID NO: 27). 44 mice were born by microinjection and in vivo transplantation of 446 fertilized eggs, and the birth rate was 9.86%; through the identification of PCR genotypes, 4F 0 mice have homologous recombination, and the positive rate is 9.1%. Positive F0 mice were identified and mated with wild-type C57BL/6N mice to obtain F1 mice.

The genotype identification method of F1 mouse generation is the same as that of F0 mouse generation, the PCR identification of the positive F1 mouse generation of homologous recombination is further carried out, the Southern blot detection of genome is further carried out, the probe binding sites are shown as 5' probe and EGFP probe in figure 2A, and the restriction enzyme cutting sites are shown as BspHI and BamHI in figure 2A. After the genomic DNA was digested with BspHI, the DNA was developed by hybridization with a 5' probe, and the DNA with a band of 9.5kb was found to be the wild type and the DNA with a band of 4.7kb was found to be the recombinant mouse. The genomic DNA was digested with BamHI and hybridized with EGFP probe, and only the 6.7kb band was found to be a positive F1 generation mouse with correct recombination and no random insertions. The Southern blot results showed that the mice of F1 generation were positive in Nos. 1 to 4, the recombinant mice with random inserts in Nos. 5 to 7, and the wild-type mice in No. 8 (FIG. 3A). The primer design sites for further identifying the genotype of the F1 mouse are shown as P3F/P3R in FIG. 2A, and the wild-type gene and the flap-EGFP knock-in allele type can respectively obtain products of 550bp and 1338bp by amplification with the primers P3F/P3R. As shown in FIG. 3B, only the 550bp product was that of the wild type mouse (+/+); only 1338bp of amplification product is homozygous mice (T/T) expressing NPC1L1-FLAG-EGFP fusion protein (NPC1L1-FG for short).

Example 2 application of NPC1L1-FLAG-EGFP mouse model

After the mice were anesthetized by inhalation of 3.5% isoflurane, the small intestine tissue was rapidly removed and divided into 6 equal portions (S1: duodenum, S2-4: jejunum, S5-6: ileum). 0.5cm of each section was cut out, immersed in 4% neutral paraformaldehyde, and fixed at 4 ℃ overnight. After washing with PBS, the tissues were dehydrated overnight in 15% and 30% sucrose solution in sequence, then embedded in o.c.t. and frozen in liquid nitrogen. Sections 8 μm thick were incubated at 55 ℃ for 20min and then mounted after staining with 10 μ g/ml DAPI in PBS for 5 min. The fluorescence image was captured using a fluorescence microscope equipped with a digital camera (Olympus BX53/DP 80).

The distribution of the NPC1L1-FLAG-EGFP protein is observed under a fluorescence microscope. Compared with +/+ mice, T/T mice exhibited significant green fluorescence from villous epithelial cells in duodenum, jejunum, and ileum, clearly delineating villous brush border membranes, and no significant green fluorescence within crypts (fig. 4). The distribution of green fluorescence is consistent with the reported distribution of NPC1L1 protein, and the EGFP gene knock-in can accurately track the expression and distribution of endogenous NPC1L1 protein.

In order to observe whether FLAG and EGFP knockin affects the expression of NPC1L1 protein, tissues such as liver, kidney, stomach, gall bladder and small intestine of +/+ and T/T mice are collected, and the expression of NPC1L1-FLAG-EGFP protein is detected by Western blot. Using anti-FLAG antibodies, the NPC1L1-FLAG-EGFP protein level was found to be undetectable in the liver, kidney, stomach and gall bladder of two mice, while the protein content of approximately 250kD in the small intestine of T/T mice was evident (FIG. 5A) and was more abundant in S3-5 (FIG. 5B). This indicates that the NPC1L1-FLAG-EGFP fusion protein is expressed in all segments of the T/T mouse small intestine and that the protein abundance is highest in the jejunum and ileum, consistent with the previously reported NPC1L1 profile.

Mice were fed with a 1.25% high cholesterol diet (D12108C, Research Diets, New Brunswick, NJ) for 3 weeks. Plasma was collected and lipids were extracted from liver and jejunum with acetone for colorimetric determination of total cholesterol and triglyceride concentrations. There were no differences in body weight, food intake, blood glucose levels between wild type and homozygous mice on the high cholesterol diet (fig. 6A-C). The morphology of the small intestine and liver in T/T mice was also similar to that of +/+ mice (FIG. 6F, I-J).

Plasma total cholesterol levels were significantly increased compared to the normal diet under the condition of High Cholesterol Diet (HCD) for 3 weeks, but there was no difference between the two genotypes (FIG. 6C). Although plasma triglyceride levels were similar in +/+ and T/T mice, there was no significant change compared to food control (FIG. 6E). In addition, no differences in intestinal and hepatic total cholesterol and triglyceride levels were observed between the two mouse strains (fig. 6G-H, K-L). These results strongly suggest that the C-terminal pair of the FLAG-EGFP protein does not affect the activity of the endogenous NPC1L1 protein.

To determine whether the EGFP tag could be used to track NPC1L1 internalization, cholesterol-induced NPC1L 1-positive vesicle trafficking needs to be further investigated. To this end, the appropriate section of intestine and sample collection time are first determined for vesicle visualization. As shown in FIG. 7A, after 200. mu.l of ink was administered by gavage, the ink reached S4 at 5 minutes, S5 at 15 minutes, and S6 at 30 minutes, respectively. Thus, after an overnight fast, T/T mice were given 200 μ l corn oil containing 4-40mg/ml cholesterol for 15 minutes or 40mg/ml cholesterol for 0-60 minutes by oral gavage. At the designated time, jejunal (S4) tissues were collected to prepare frozen sections. NPC1L1 positive vesicles were observed directly under a fluorescent microscope after DAPI staining. As shown in fig. 7C, NPC1L1 positive vesicles below the brush border membrane were visible as early as 5 minutes after 40mg/ml cholesterol feeding. The number of vesicles increased to a peak at 15 minutes and decreased greatly at 60 minutes (fig. 7E). As expected, vesicle formation increased with increasing cholesterol dose and the most abundant signal was reached at a concentration of 20mg/ml (fig. 7D). However, larger vesicles were visible at a concentration of 40mg/ml (FIG. 5B).

We further determined whether vesicles are NPC1L1 specific by using the NPC1L1 specific inhibitor Ezetimibe (Ezetimibe). It was found that pretreatment of 10mg/kg Ezetimibe for 5 days by gastric tube feeding significantly reduced cholesterol-induced brush border subconjunctival vesicle formation after 30 minutes of cholesterol gavage (FIG. 8).

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.

Sequence listing

<110> China people liberation army navy military medical university

Zhuxianyi Memorial Hospital, Tianjin Medical University (Tianjin Medical University Metabolic Disease Hospital, Tianjin Metabolic Disease Control Center)

<120> construction method of transgenic non-human animal and application thereof

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Claims (10)

1. A method for constructing a transgenic non-human animal, which comprises inserting a reporter gene and/or a gene encoding a tag protein into the 3' noncoding region of the NPC1L1 gene of the transgenic non-human animal.

2. The method of claim 1, wherein the insertion site is located between the end of the reading frame of the NPC1L1 protein and the 3' UTR, preferably between the end of the reading frame of the NPC1L1 protein and the stop codon.

3. The construction method according to claim 1, wherein the non-human animal expresses a fusion protein of NPC1L1 protein-tag protein-reporter gene encoding protein, preferably, NPC1L1 protein and tag protein are linked, and tag protein and reporter gene encoding protein are linked by a connecting peptide, the connecting peptide has the sequence shown in SEQ ID NO: shown at 30.

4. The method of claim 1, wherein the reporter gene is selected from CAT, hGH, SEAP, GFP, EGFP, β -Gal, TdTomato, mCherry, and firefly luciferase genes, preferably the reporter gene is EGFP, and the amino acid sequence of the EGFP protein is as shown in SEQ ID NO: shown at 31.

5. The method for constructing a recombinant expression vector of claim 1, wherein the tag protein is selected from Myc tag protein, FLAG tag protein, His6 tag protein, T7 tag protein, V5 tag protein, HA tag protein and GST tag protein, preferably, the tag protein is FLAG tag protein, and more preferably, the amino acid sequence of the FLAG tag protein is as shown in SEQ ID NO: shown at 21.

6. The construction method according to claim 1, characterized in that the construction method includes constructing a transgenic non-human animal using sgRNA and/or targeting vectors, wherein,

the sequence of the sgRNA target site is shown in SEQ ID NO: 13-20, preferably, the sgRNA sequence is as shown in SEQ ID NO: 5 to 12;

the targeting vector comprises a5 'arm, a 3' arm and a donor DNA sequence, wherein the donor DNA sequence comprises a nucleotide sequence for coding a tag protein and a reporter gene;

preferably, the 5' arm is as set forth in SEQ ID NO: as shown in figure 3, the first and second,

preferably, the 3' arm is as set forth in SEQ ID NO: as shown at 28, the flow of the gas,

preferably, the donor DNA sequence is as set forth in SEQ ID NO: as shown at 29.

7. The method of any one of claims 1-6, wherein the non-human animal is a non-human mammal, preferably a rat or a mouse.

8. The sgRNA targeting the NPC1L1 gene is characterized in that the sequence of the target site of the sgRNA is shown in SEQ ID NO: 13-20, wherein the sgRNA sequence is shown in SEQ ID NO: 5 to 12.

9. A targeting vector comprising a5 'arm, a 3' arm, and a donor DNA sequence, said donor DNA sequence comprising a nucleotide sequence encoding a tag protein and a reporter gene;

preferably, the 5' arm is as set forth in SEQ ID NO: as shown in figure 3, the first and second,

preferably, the 3' arm is as set forth in SEQ ID NO: as shown at 28, the flow of the gas,

preferably, the donor DNA sequence is as set forth in SEQ ID NO: as shown at 29.

10. Use of the non-human animal or its progeny obtained by the construction method according to any one of claims 1 to 7, the sgRNA according to claim 8, or the targeting vector according to claim 9 for in vivo tracking of NPC1L1 protein and for the preparation of a medicament for the treatment or prevention of cholesterol-related diseases.

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