CN116769834A - Method for constructing adipose tissue-specific STAU1 knockout mouse model by Myf5-Cre - Google Patents
Method for constructing adipose tissue-specific STAU1 knockout mouse model by Myf5-Cre Download PDFInfo
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Abstract
The invention discloses a method for constructing an adipose tissue-specific STAU1 knockout mouse model by Myf5-Cre, which comprises the steps of designing targeted sgRNA based on a CRISPR/Cas9 system according to the 3-6 exon sequence of a STAU1 gene part of the mouse, carrying out in vitro transcription on mRNA of the sgRNA and Cas9 nuclease, carrying out microinjection into sperm eggs of the mouse, and transplanting fertilized egg embryos after injection into a pseudopregnant female mouse to obtain an F0 mouse; extracting the DNA of the rat tail blood of the F0 generation, sequencing the amplified product by PCR, and identifying the genotype to obtain a positive F0 generation rat; mating the F0 mice with wild mice respectively to obtain heterozygote mice Fl generation, and then carrying out rat tail identification; the F2 generation homozygous mice, namely STAU1/myf5 knockout mice model, are obtained by crossing Fl generation mice. The invention aims to construct a STAU1/myf5 knockout animal model to explore the role of STAU1 in the differentiation process of brown adipocytes, and explore the heat generation mechanism of STAU1 for inhibiting BAT through methods such as weight, glucose tolerance, insulin release and the like of mice.
Description
Technical Field
The invention belongs to the technical field of animal experiment model construction, and particularly relates to a method for constructing an adipose tissue-specific STAU1 knockout mouse model by Myf 5-Cre.
Background
Obesity has become a global chronic metabolic disease, which can lead to type 2 diabetes, cardiovascular and cerebrovascular diseases, fatty liver, and serious health hazard to humans. Adipose tissue in mammals is believed to include mainly white adipose tissue with energy storage function (White adipose tissue, WAT) and brown adipose tissue with heat and energy production (Brown adipose tissue, BAT). In recent years, it has been found that when the body is exposed to cold or drug-like stimuli, some multi-atrial uncoupling protein 1 (Uncoupling protein, UCP 1) positive adipocytes appear in WAT, which function similarly to brown adipocytes. Such adipocytes with white and brown adipocyte intermediate morphology are called beige adipocytes. Such beige adipocytes can be induced in WAT reservoirs by complex processes and exhibit similar thermogenesis to BAT upon stimulation. It has been shown that mammalian adipocytes are derived mainly from precursors of the neural kurtosis or mesoderm, wherein the neural kurtosis can differentiate directly into mature adipocytes, whereas precursors from mesoderm can differentiate into different types of precursor cells with specific differentiation capabilities, and thus into different types of adipocytes or muscle cells. Brown adipocytes develop from paraxial mesoderm, where Myf5 expressing mesoderm progenitor cells can differentiate into muscle or brown adipose precursor cells. Brown fat precursor cells can gradually differentiate into brown fat cells after being regulated by Ehmt1, prdm16, C/EBPS, ews and other factors. However, little is known about the developmental origin of beige adipocytes, and previous studies reported that classical brown fat and skeletal muscle cells were derived from Myf5 myogenic precursor cell lineages, and white and beige adipocytes were derived from Myf5 precursor cells, knowing the differences in developmental profile of brown and beige fat provided more guidance for the treatment of obesity and metabolic diseases.
Abnormal adipocyte differentiation is the pathophysiological basis for obesity, in which many transcription factors are involved, and RNA binding proteins also regulate adipocyte differentiation at post-transcriptional levels, where STAU1 is involved in the regulation of adipose tissue and glycolipid metabolism. Preliminary studies have found that STAU1 may be able to inhibit adipose tissue thermogenesis by affecting Ucp1 and Prdm16 thermogenesis gene expression, and that the specific mechanism is not yet defined. In the invention, STAU1 deletion is observed in STAU1/adipo mice, which possibly promotes the heat production of the mice, but because the expression quantity of adipoQ in BAT is lower and the knockout efficiency is limited, at present, the gene knockout type obesity animal model for scientific research is very limited, and the market does not have a stable hereditary gene knockout type obesity model mouse, which is required to be cultivated and reformed in the acquired, has longer modeling time, low modeling success rate and high death rate. Thus, there is a need for a method of constructing a adipose tissue-specific STAU1 knockout mouse model by Myf 5-Cre.
Disclosure of Invention
The invention provides a method for constructing an adipose tissue-specific STAU1 knockout mouse model by screening Myf5-Cre of an optimal modeling scheme through evaluating model establishment conditions by adopting methods such as survival conditions, physical sign and motion state observation, flow cytometry, wester Blot and PCR.
The invention comprises the following steps:
1) sgRNA target design: according to the sequence set forth in SEQ ID NO:1, and the sequence of the first sgRNA target is shown as SEQ ID NO:2, the second sgRNA target sequence is shown as SEQ ID NO:3 is shown in the figure; the third sgRNA target sequence is shown as SEQ ID NO:4, the fourth sgRNA target sequence is shown in SEQ ID NO:5 is shown in the figure;
2) Construction of CRISPER/Cas9 recombinant plasmid: using primers as set forth in SEQ ID NO:6, performing PCR amplification to obtain a CRISPER/Cas9 recombinant plasmid;
3) Amplifying the CRISPER/Cas9 recombinant plasmid DNA, then carrying out enzyme tangentially, carrying out in vitro transcription, microinjection into sperm eggs of mice, and transplanting the fertilized egg embryos after injection into pseudopregnant female mice to obtain F0 mice;
4) Mating the F0 generation mice with the wild type mice with different polarities, and identifying offspring genotypes to obtain F1 generation heterozygous gene knockout mice;
5) By inbreeding the F1 generation heterozygous gene knockout mice, primers SEQ ID NO:7, obtaining the F2 generation homozygous gene knockout mouse by PCR screening genotype identification, namely the STAU1/myf5 mouse model.
Further, the PCR amplification system is
Wherein the PCR reaction program is 94 ℃, 3min,94 ℃, 30s,60 ℃, 35s,72 ℃ and 5min for 38 cycles.
Further, the C57/BL6 female mice with the age of 4-6 weeks are injected with 10IU of pregnant mare serum gonadotropin, 10IU of human chorionic gonadotropin is injected 48 hours later, and the mice are matched with C57/BL6 male mice in a cage and are collected with vaginal suppositories; preparation of recipient female mouse for male mouse with reproductive function of 4 weeks old or more
Further, fertilized eggs which survive the injection are transferred to an M16 culture medium, placed in a saturated humidity incubator at 37 ℃ and 5% CO2 and 5% O2, and transplanted after 30-60 min.
Further, viable fertilized eggs were cultured after ovarian microinjection of C57/BL6 female mice selected for pessary.
A model of obesity in mice caused by gene defect is prepared by selecting Myf5 promoter for mouse with fat tissue specific STAU1 knocked out, and constructing STAU1/Myf5 as model of mouse.
The meaning of the invention is as follows:
according to the invention, myf5 promoter is selected, target sgRNA is designed based on CRISPR/Cas9 system according to the 3-6 exon sequence of the STAU1 gene part of the mouse, and STAU1/Myf5 knockout animal model is constructed through multiple hybridization breeding, so that the change of the heat production function of the mouse after STAU1 is knocked out in brown adipocytes is explored. Modeling conditions are set, the model is evaluated through methods such as survival conditions, body surface signs, metabolic levels and the like of mice, and an optimal STAU1/myf5 knockout animal model scheme is screened, so that a reliable and simple animal model is provided for further research on metabolic diseases and obesity gene therapy.
Drawings
FIG. 1 is a schematic diagram of the identification of a STAU1 specific knockout mouse gene in an embodiment of the invention;
FIG. 2 is a graph showing the results of gene testing after normal mating and reproduction of STAU1 idiotype knockout mice in the present invention;
fig. 3 is a dnagarer specification.
FIG. 4 shows that the body weight gain curve shows that STAU1/myf5 mice are significantly higher than control mice after 6 weeks of high fat diet, whereas the average body weight of STAU1/myf5 mice was about 4 grams lighter than control mice by 17 weeks of high fat diet, and the difference was statistically significant.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1 construction of CRISPER-Cas9 recombinant plasmid
sgRNA target design
1) sgRNA target design: according to the sequence set forth in SEQ ID NO:1, and the sequence of the first sgRNA target is shown as SEQ ID NO:2, the second sgRNA target sequence is shown as SEQ ID NO:3 is shown in the figure; the third sgRNA target sequence is shown as SEQ ID NO:4, the fourth sgRNA target sequence is shown in SEQ ID NO:5 is shown in the figure;
2) Construction of CRISPER/Cas9 recombinant plasmid: using primers as set forth in SEQ ID NO:6, performing PCR amplification to obtain a CRISPER/Cas9 recombinant plasmid;
3) Amplifying the CRISPER/Cas9 recombinant plasmid DNA, then carrying out enzyme tangentially, carrying out in vitro transcription, microinjection into sperm eggs of mice, and transplanting the fertilized egg embryos after injection into pseudopregnant female mice to obtain F0 mice;
4) Mating the F0 generation mice with the wild type mice with different polarities, and identifying offspring genotypes to obtain F1 generation heterozygous gene knockout mice;
5) By inbreeding the F1 generation heterozygous gene knockout mice, primers SEQ ID NO:7, obtaining the F2 generation homozygous gene knockout mouse by PCR screening genotype identification, namely the STAU1/myf5 mouse model.
CRISPR-Cas 9 system for STAU1/myf5 gene knockout, wherein the sgRNA action site in the CRISPR-Cas 9 system is positioned on exons 3-6 of STAU1 gene, and the DNA sequence of the sgRNA action site is as follows:
Exon3:5'—ATACTTTTACCCATTTCCAGTCCCACCTTTACTCTACCAAGTTGA GCTCTCCGTGGGCGGACAGCAGTTTAATGGGAAAGGAAAGATGAGACCAC CCGTGAAACACGATGCCCCTGCCCGTGCGCTGAGGACTCTGCAGAGTGAA CCCCTGCCAGAAAGGTTGGAG—3'
Exon4:5'—GTAAATGGAAGAGAAGCAGAGGAAGAAAACCTCAATAAATCGG AAATAAGCCAAGTGTTTGAAATTGCGCTGAAGCGGAATTTGCCTGTGAATT TTGAG—3'
Exon5:5'—GTGGCCCGGGAGAGTGGCCCACCACACATGAAGAACTTTGTGA CCAGGGTTTCAGTTGGGGAATTTGTAGGGGAAGGAGAAGGGAAAAGCAAGAAGATCTCCAAGAAGAATGCGGCCAGGGCTGTTCTGGAGCAGCTTAGGAGGCTGCCACCCCTCCCTGCTGTGGAGCGAGTGAAGCCCAGAATCAAGAAGAAAAGTCAGCCCACCTGCAAG—3'
Exon6:5'—CTACAGACAGCCCCGGATTATGGCCAAGGGATGAATCCTATT AGTAGACTTGCACAGATCCAGCAGGCAAAAAAGGAGAAGGAGCCAGAGT ACATGCTCCTTACAGAACGAGGTCTTCCACGTCGCAGGGAGTTTGTGATGC AG—3'
in this embodiment, the PCR amplification system is
Wherein the PCR reaction program is 94 ℃, 3min,94 ℃, 30s,60 ℃, 35s,72 ℃ and 5min for 38 cycles.
The PCR specific primer is SEQ ID NO:6
Primers for Myf5-Cre-WT PCR:
Myf5-Cre-P3:5'-AACCAGAGACTCCCCAAGGT-3’
Myf5-Cre-P2:5'-CGGCTCTTAAAGCAATGGTC-3'
Preparation of gene fragment for microinjection
Plasmid DNA was amplified, digested and linearized, purified by gel recovery, and then dissolved in sterile TE (5mmolLTris,0.1mmolLEDTA,pH 7.4) buffer, diluted and quantified to 5ng/uL, and stored at-20℃until injection was awaited.
Preparation of donor mice, ligating male mice, and recipient mice
The illumination time of the animal feeding room was adjusted to 5:0019:00, and the night was from 19:00 to 5:00 the next day.
Injecting 10IUPMSG into C57/BL6 female mice with the age of 4-6 weeks, injecting 10IU h CG after 48h, mating with C57/BL6 male mice in a cage, and collecting mice with vaginal suppositories; anesthesia is carried out on KM male mice with reproductive function of more than 4 weeks old, vas deferens are cut off through surgery, after suturing, the mice are kept for 4 weeks, and the mice are caged with a female mouse to prepare a recipient female mouse; selecting KM female mice with age of more than 4 weeks, and combining with ligature male mice, collecting female mice with thrombus on the next day, and performing embryo transfer.
Fertilized egg collection and treatment
The C57/BL6 female mice with pessaries were humanly sacrificed, laparotosed, and ovaries and part of uterine horns were removed. The removed tissue was washed with M2, placed in a new M2 solution, and the oviduct was dissected with a sharp instrument under a microscope to allow fertilized eggs to flow out of the oviduct. The fertilized eggs with granulosa cells were transferred to 1mg/mL hyaluronidase by a pipette to digest and remove granulosa cells. After washing in M2 operating fluid for several times, 2 fertilized eggs with good prokaryotes and morphology are selected, and transferred to M16 culture fluid to be placed in a saturated humidity incubator with 5% CO2 and 5% O2 at 37 ℃ for standby.
Microinjection
Taking 20-30 fertilized eggs in an injection tray, injecting larger procaryotes (male procaryotes), and calculating successful injection when the procaryote volume is obviously increased by 1/3 of the original procaryote volume. If fertilized egg procaryon is not obviously increased, the exogenous DNA injection is not injected into the procaryon, and the injection is ineffective. After the injection is completed, the fertilized eggs which survive the injection are transferred into an M16 culture solution, placed in a saturated humidity incubator for 30-60 min at 37 ℃ and 5% CO2, 5% O2, and transplanted.
Embryo transfer
Taking a recipient mouse with the same day of thrombus, taking out the ovary at the abdomen side by surgical operation after anesthesia, tearing the ovary serosa by forceps, finding the ampulla of the oviduct, inserting a transplanting needle with about 25 embryo after injection into the oviduct, transplanting the embryo into the oviduct, and then sending the ovary and the oviduct back into the recipient mouse body for suturing.
Acquisition and genotyping of BAT knockout STAU1 Gene mice
Transplanting the fertilized eggs which are cultured and survived after microinjection into a pseudopregnant female mouse body through the umbrella end of the oviduct, extracting DNA (deoxyribonucleic acid) from the birth of the mouse after 19-21 d, and performing PCR (polymerase chain reaction) and sequencing to identify and screen genotypes to obtain F0-generation mice; mating the F0 generation mice with the wild type mice with different polarities, and identifying offspring genotypes to obtain F1 generation heterozygous gene knockout mice; and F1 generation heterozygous mice are subjected to male-female inbreeding, and offspring genotype identification is carried out to obtain F2 generation homozygous knockout mice. Primer SEQ ID NO: as shown in figure 7 of the drawings,
5’-CATTTCCAAAAGACGTCACCCTTA-3’；
5’-AGTAGCTGACACATGCTATGTACG-3’。
therefore, the Myf5 promoter is selected, STAU1/Myf5 is to be constructed as a main animal model to explore the function of STAU1 in the differentiation and maturation process of brown fat cells, and the heat generation mechanism of STAU1 for inhibiting BAT is explored, wherein the brown fat cells originate from primitive intestinal mesoderm mesenchymal stem cells and gradually differentiate into mature brown fat cells under the drive of a transcription factor Myf5, so that the expression of Cre enzyme can be specifically started in brown fat tissues, and STAU1 is knocked out. The invention adopts transgenic mouse technology to prepare tissue-specific transgenic mouse model by microinjection of constructed widely expressed promoter-lox-Stop-lox-transgenic vector, the transgene is not expressed under normal condition, only after hybridization with corresponding tissue-specific expression Cre/Creet 2 mouse, stop termination sequence is removed in specific tissue, thus achieving the purpose of specific expression of transgene in inductivity/specific tissue.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (6)
1. A method for constructing an adipose tissue-specific stu 1 knockout mouse model by Myf5-Cre, comprising the steps of:
1) sgRNA target design: according to the sequence set forth in SEQ ID NO:1, and the sequence of the first sgRNA target is shown as SEQ ID NO:2, the second sgRNA target sequence is shown as SEQ ID NO:3 is shown in the figure; the third sgRNA target sequence is shown as SEQ ID NO:4, the fourth sgRNA target sequence is shown in SEQ ID NO:5 is shown in the figure;
2) Construction of CRISPER/Cas9 recombinant plasmid: using primers as set forth in SEQ ID NO:6, performing PCR amplification to obtain a CRISPER/Cas9 recombinant plasmid;
3) Amplifying the CRISPER/Cas9 recombinant plasmid DNA, then carrying out enzyme tangentially, carrying out in vitro transcription, microinjection into sperm eggs of mice, and transplanting the fertilized egg embryos after injection into pseudopregnant female mice to obtain F0 mice;
4) Mating the F0 generation mice with the wild type mice with different polarities, and identifying offspring genotypes to obtain F1 generation heterozygous gene knockout mice;
5) By inbreeding the F1 generation heterozygous gene knockout mice, primers SEQ ID NO:7, obtaining the F2 generation homozygous gene knockout mouse by PCR screening genotype identification, namely the STAU1/myf5 mouse model.
2. The method for constructing a STAU1/myf5 knockout mouse model according to claim 1, wherein the PCR amplification system is
Wherein the PCR reaction program is 94 ℃, 3min,94 ℃, 30s,60 ℃, 35s,72 ℃ and 5min for 38 cycles.
3. The method for constructing an obese mouse model according to claim 1, wherein the method for preparing the recipient master mouse comprises: injecting 10IU of pregnant mare serum gonadotropin into a C57/BL6 female mouse with the age of 4-6 weeks, injecting 10IU of human chorionic gonadotropin after 48 hours, mating with a C57/BL6 male mouse in a cage, and collecting mice with vaginal suppositories; the recipient female mice were prepared by cage-joining male mice with reproductive function and female mice over 4 weeks of age.
4. The method for constructing an obese mouse model according to claim 1, wherein the fertilized eggs which survive the injection in the step B are transferred to an M16 culture medium, placed in a saturated humidity incubator at 37℃with 5% CO2 and 5% O2, and transplanted after 30 to 60 minutes.
5. The method of claim 1, wherein the viable fertilized eggs are cultured after ovarian microinjection of C57/BL6 female mice selected from pessary.
6. A mouse obesity model caused by gene defect is characterized in that for a mouse with fat tissue specificity STAU1 knocked out, a Myf5 promoter is selected, and STAU1/Myf5 is designed to be constructed as a mouse modeling model.
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