CN113491255A - Construction method and application of obese type II diabetic zebra fish model - Google Patents
Construction method and application of obese type II diabetic zebra fish model Download PDFInfo
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Abstract
The invention discloses a construction method and application of an obese type II diabetic zebra fish model, wherein the obese type II diabetic zebra fish model with stable hereditary character is constructed by knocking out mc4r gene, the constructed zebra fish model has obese and type II diabetic symptoms at the same time, the clinical symptoms of the obese and type II diabetes can be stably inherited, an animal model with stable source can be obtained by natural breeding of animals by one-time construction, compared with a simple physiological induction model, the construction method is rapid and efficient, and the experimental efficiency of the animal model can be effectively improved and the experimental cost can be effectively reduced.
Description
Technical Field
The invention relates to the technical field of animal models, in particular to a construction method and application of an obese type II diabetic zebra fish model.
Background
With the increase of daily living standard, obesity has become a serious problem endangering human health. Obesity is a metabolic syndrome caused by excessive fat tissue growth due to excessive energy intake, which can lead not only to insulin resistance but also to type II diabetes in patients, and thus many obese patients are also type II diabetes patients.
In the past years, rodent animals such as rabbits, rats and mice are generally used as drug evaluation models for clinical treatment of obesity and type II diabetes and animal models for researching pathogenesis of the obesity and type II diabetes, a common method for constructing the animal models is obtained by feeding wild animals with high sugar and high fat for induction, but the problems of long model construction time, high cost, high feeding space requirement, unstable genetic traits and the like exist, each batch of animal models need to be reconstructed after use, and even if a transgenic technology is adopted to obtain a stable genetic mutation system, the defects of complex operation, long period and high economic cost exist.
And zebra fish is common tropical fish, has high genetic homology of 87% with human and has high biological similarity with human, which means that experimental results are applicable to human bodies in most cases. They are small in size, less than 5cm in length, do not occupy space, and are more convenient to raise than rats or mice. The zebra fish has short breeding cycle, no season for mating, more spawning, rapid embryo development, and can complete the construction of internal organs of the body after 24 hours after fertilization, and the embryo is transparent and easy to observe.
And the zebra fish, which is a model organism such as a small mouse, has obvious advantages in pharmacological experiments of the slow-acting drugs. Firstly, the establishment time of the zebra fish chronic disease model is short. Compared with a mouse and the like which need to construct disease models such as diabetes and the like for months, the zebra fish can form a pathological model with typical disease characteristics only for a few days, and time, expenditure and energy can be greatly saved. Secondly, the zebra fish model for establishing the chronic disease has short treatment period. Similarly, compared with model animals such as mice, zebra fish can cause obvious pathological state change only by taking a plurality of days of administration, and the experimental period can be effectively shortened. Meanwhile, the zebra fish can also simulate the characteristics of complicated slow disease medication. And thirdly, the zebra fish breeding space is low in cost, compared with model animals such as mice, the zebra fish breeding condition is relatively simple, the occupied area is small, and the space required by case model construction is small. In addition, the zebrafish is simple in administration mode, the medicine can be directly dissolved in water, and the zebrafish can be absorbed through skin, gills, a digestive system and the like.
However, in the current research, few researches are conducted to construct an obese type II diabetic zebra fish model by a gene knock-out method.
Disclosure of Invention
Therefore, based on the above background, the present invention provides a method for constructing an obese type II diabetic zebra fish model, which constructs an obese or type II diabetic zebra fish model with stable genetic characteristics by knocking out mc4r gene, and can greatly reduce the cost pressure caused by the construction of current popular rodent models such as mouse models of small, rat, and rabbit.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a construction method of an obese type II diabetic zebra fish model comprises the following construction steps:
1) obtaining a wild zebra fish egg:
placing the male and female of the normally cultured wild zebra fish in a zebra fish mating tank according to a certain proportion after the lamp is turned on, respectively placing the male and female on two sides of a baffle, standing for 1.5-4 hours, then removing the baffle, and collecting fertilized eggs settled at the bottom of the tank in time after 0.5-3 hours;
2) constructing a gene knockout zebra fish:
firstly, carrying out enzyme digestion linearization on the pCS2-Cas9 plasmid by using Xba I, purifying and transcribing an enzyme digestion product into Cas9mRNA in vitro, and then storing at-60-95 ℃;
② selecting a mc4r gene candidate Cas9 target site, respectively synthesizing an upstream primer and a downstream primer,
wherein the sequence of the upstream primer is as follows:
AATTAATACGACTCACTATAGGCTGACCAGGCCGAGCGTGGTTTTAGAGCTAGAAATAGC(SEQIDNO.1)；
the sequence of the downstream primer is as follows:
GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQIDNO.2)；
amplifying an upstream primer and a downstream primer by using PCR to obtain an sgRNA in-vitro transcription template, transcribing the sgRNA in vitro to synthesize the sgRNA, and storing the sgRNA at a temperature of between 60 ℃ below zero and 95 ℃;
③ 0.5-5 mul of Cas9mRNA and sgRNA are evenly mixed to form a mixed solution, and the mixed solution is stored on ice;
dividing the fertilized eggs of the zebra fish in the step 1) into an injection group and a control group, and microinjecting 1nl of mixed solution to the interface of yolk and cells of the fertilized egg cells of 1-2 generations in the injection group;
culturing the zebra fish fertilized egg cells of the injection group and the control group in an incubator at 25-31 ℃, and replacing the culture solution at night on the day of injection;
3) obtaining stable genetic zebra fish homozygous lines:
culturing embryos which are split and developed by the injected group oosperms to adult individuals, carrying out tail shearing identification on the adult individuals, and screening a mutant F0 generation;
② crossing adult zebra fish F0 with wild zebra fish to obtain heterozygous F1 generation;
and thirdly, selfing the adult heterozygous zebra fish F1, and screening to obtain the homozygous zebra fish F2.
4) Construction of obese type II diabetes zebra fish model
Firstly, normally feeding the homozygous zebra fish F2 screened in the step 3) for 3 months by using the fairy shrimp as a feed;
secondly, after the cooked yolk added with 10% cholesterol is used as feed to feed for 12-18 days, the blood sugar of the zebra fish of the control group and the zebra fish cultured by the injection group is detected, the blood sugar content of the zebra fish of the injection group after 1.5 hours and 8 hours after the meal is more than 1.5 times of the blood sugar content of the zebra fish of the control group, and the establishment success of the obese type II diabetes zebra fish model can be judged.
Further, the placing ratio of the male and female wild zebra fish in the step 1) is 1:2 or 1: 1.
Further, the preparation method of the culture solution in the step 2) comprises the following steps: 200ml of KC solution with the concentration of 0.5 wt%, 200ml of CaCl2 solution with the concentration of 1 wt% and 200ml of NaHCO3 solution with the concentration of 0.25 wt% are mixed, 70g of solid NaCl and a proper amount of ddH2O are added to the mixture to be dissolved, the volume is kept to 20L, and then 26ml of methylene blue solution is added.
Further, the concentration of Cas9mRNA before mixing in the third step of step 2) was 600ng/μ l, and the concentration of sgRNA was 300ng/μ l.
The invention also provides application of the obese type II diabetic zebra fish model constructed by the model in the research of diabetes mechanism or in screening and evaluating medicines for treating type II diabetes.
Compared with the prior art, the invention has the advantages that: the obese type II diabetes zebra fish model with stable hereditary character is constructed by knocking out the mc4r gene, the constructed zebra fish model has the symptoms of obesity and type II diabetes at the same time, the clinical symptoms of obesity and type II diabetes can be stably inherited, and the animal model with stable source can be obtained by natural breeding of animals through one-time construction;
the model features and the genetic characters constructed by the invention are stable, can be used as research materials for researching the pathological mechanism of obesity and type II diabetes, can also be used for screening and evaluating a medicament for clinically treating obesity and type II diabetes, and have important significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a map of a pCS2-Cas9 plasmid of the present invention;
FIG. 2 is a flow chart of screening zebrafish mc4r gene knockout homozygous lines of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: a construction method of an obese type II diabetic zebra fish model comprises the following construction steps:
1) obtaining a wild zebra fish egg:
placing the normally cultured wild zebra fish in a zebra fish mating tank according to the proportion of 1:1 or 1:2 after the lamp is turned on, respectively placing the male and female in the two sides of the baffle, standing for 2 hours, then removing the baffle, and collecting fertilized eggs settled at the bottom of the tank in time after 1 hour;
2) constructing a gene knockout zebra fish:
firstly, carrying out enzyme digestion linearization on the pCS2-Cas9 plasmid by using Xba I, purifying and transcribing an enzyme digestion product into Cas9mRNA in vitro, and then storing at-80 ℃;
secondly, selecting a candidate Cas9 target site of the mc4r gene by using a CHOPCHOP online website (https:// chlorophtop. rc. fas. harvard. edu /), synthesizing an upstream primer and a downstream primer respectively,
wherein the sequence of the upstream primer is as follows:
AATTAATACGACTCACTATAGGCTGACCAGGCCGAGCGTGGTTTTAGAGCTAGAAATAGC(SEQIDNO.1)；
the sequence of the downstream primer is as follows:
GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQIDNO.2)；
amplifying an upstream primer and a downstream primer by using PCR to obtain an sgRNA in-vitro transcription template, transcribing the sgRNA in vitro to synthesize the sgRNA, and storing the sgRNA at-80 ℃;
③ evenly mixing 1 microlitre of Cas9mRNA (concentration: 600 ng/. mu.l) and sgRNA (300 ng/. mu.l) to obtain a mixed solution, and placing the mixed solution on ice for storage;
dividing the fertilized eggs of the zebra fish in the step 1) into an injection group and a control group, and microinjecting 1nl of mixed solution (about 1/10 of the fertilized eggs) to the interface of yolk and cells of the fertilized egg cells of 1-2 generations of the injection group;
culturing the fertilized egg cells of zebra fish in 28.5 deg.C incubator, replacing culture solution at night on the day of injection,
the preparation method of the culture solution comprises the following steps: 200ml of KC solution with the concentration of 0.5 wt%, 200ml of CaCl2 solution with the concentration of 1 wt% and 200ml of NaHCO3 solution with the concentration of 0.25 wt% are mixed, 70g of solid NaCl and a proper amount of ddH2O are added to the mixture to be dissolved, the volume is kept to 20L, and then 26ml of methylene blue solution is added.
3) Obtaining stable genetic zebra fish homozygous lines: (see the attached figure 2 for details)
Culturing embryos which are split and developed by the injected group oosperms to adult individuals, carrying out tail shearing identification on the adult individuals, and screening a mutant F0 generation;
② crossing adult zebra fish F0 with wild zebra fish to obtain heterozygous F1 generation;
and thirdly, selfing the adult heterozygous zebra fish F1, and screening to obtain the homozygous zebra fish F2.
4) Construction of obese type II diabetes zebra fish model
Firstly, normally feeding the homozygous zebra fish F2 screened in the step 3) for 3 months by using the fairy shrimp as a feed; culturing embryos of the control group of zebra fish fertilized eggs for division development into individuals, normally feeding the individuals by adopting the fairy shrimp as a feed, and taking the cultured zebra fish as a wild zebra fish;
secondly, after the cooked yolk added with 10% cholesterol is used as feed to feed for 12-18 days, the blood sugar of the zebra fish of the control group and the zebra fish cultured by the injection group is detected, the blood sugar content of the zebra fish of the injection group after 1.5 hours and 8 hours after the meal is more than 1.5 times of the blood sugar content of the zebra fish of the control group, and the establishment success of the obese type II diabetes zebra fish model can be judged.
In the step, selecting individuals in F2 generation zebra fish which is normally fed, wherein the individuals are obviously larger than wild zebra fish which is normally fed and cultured, and sequencing the selected individuals to find that the mc4r gene of the fishes is mutated, so that the obese zebra fish model can be judged to be successful; and after the fact that the fish with the mutation of the mc4r gene is fed with high sugar and high fat for 14 days by taking cooked egg yolk as a feed is determined, judging the blood sugar, and judging the success construction of the obese type II diabetic zebra fish model when the blood sugar content of the fish after 1.5 hours and 8 hours is more than 1.5 times of that of a control group.
The method comprises the following steps of (1) successfully constructing an obese type II diabetic zebra fish model (specifically, the body length and the body weight of the obese type II diabetic zebra fish model are shown in tables 1 and 2) which is obviously larger than the size of a control group zebra fish through visual observation; secondly, blood sugar content is tested by blood sugar test paper, and the blood sugar content after 1.5 hours and 8 hours (the detection value is shown in table 3) is more than 1.5 times of that of the control group.
Table 1: body weight statistics (average) for zebra fish of 3-6 months old
| Group of | Age of 3 months | 4 months old | Age of 5 months | Age of 6 months |
| Control group | 0.253g | 0.298g | 0.352g | 0.414g |
| Model set (Male) | 0.265g | 0.326g | 0.426g | 0.585g |
| Model set (female) | 0.269g | 0.332g | 0.441g | 0.625g |
Table 2: body weight statistics (average) for zebra fish of 3-6 months old
| Group of | Age of 3 months | 4 months old | Age of 5 months | Age of 6 months |
| Control group | 2.02cm | 2.53cm | 2.72cm | 2.95cm |
| Model set (Male) | 2.14cm | 2.61cm | 2.86cm | 3.12cm |
| Model set (female) | 2.18cm | 2.70cm | 2.98cm | 3.22cm |
Table 3: blood glucose level detection
| Experiment grouping | After meal 1.5h (mmol/L) | After meal 8h (mmol/L) |
| Control group (AB) | 2.82 | 2.35 |
| Model set | 4.85 | 4.30 |
The zebra fish serving as a model organism for scientific research has the advantages of high homology with human genes, small culture space, short breeding time, large number of filial generations and the like, and can be used as an ideal animal model for developing a medicament evaluation model for clinically treating obesity and type II diabetes and researching pathogenesis. Melanocortin receptor-4 (mc4r) is a peptide substance secreted by hypothalamus and capable of regulating food intake and maintaining energy homeostasis in vivo, and the zebra fish with mc4r deficiency has the phenomena of food intake increase, fertility enhancement and energy metabolism imbalance, and can have the symptoms of obesity and type II diabetes after being slightly induced.
Compared with the obese type II diabetic zebra fish model constructed by a physiological induction method (namely, the obese type II diabetic zebra fish model is fed by high-fat and high-cholesterol feed), after the normal diet is recovered for a period of time, the secretion of blood sugar and insulin can possibly recover the symptom state, the model characteristic is unstable, the symptoms have no obvious genetic characteristic, and offspring needs to be re-induced and constructed, the construction method of the obese type II diabetic zebra fish model constructed by the method of knocking out mc4r is simple in operation, the model construction power is high, the genetic character is stable, the offspring can still have the model characteristic through normal breeding, and the method can improve the experimental efficiency greatly and reduce the experimental period and the cost when the method is applied to evaluation and screening of medicines for treating type II diabetes and pathological mechanism research of diabetes.
The invention and its embodiments have been described above, without this being limitative. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (5)
1. A method for constructing an obese type II diabetic zebra fish model is characterized by comprising the following construction steps:
1) obtaining a wild zebra fish egg:
placing the male and female of the normally cultured wild zebra fish in a zebra fish mating tank according to a certain proportion after the lamp is turned on, respectively placing the male and female on two sides of a baffle, standing for 1.5-4 hours, then removing the baffle, and collecting fertilized eggs settled at the bottom of the tank in time after 0.5-3 hours;
2) constructing a gene knockout zebra fish:
firstly, carrying out enzyme digestion linearization on the pCS2-Cas9 plasmid by using Xba I, purifying and transcribing an enzyme digestion product into Cas9mRNA in vitro, and then storing at-60-95 ℃;
② selecting a mc4r gene candidate Cas9 target site, respectively synthesizing an upstream primer and a downstream primer,
wherein the sequence of the upstream primer is as follows:
AATTAATACGACTCACTATAGGCTGACCAGGCCGAGCGTGGTTTTAGAGCTAGAAATAGC(SEQ ID NO.1)；
the sequence of the downstream primer is as follows:
GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQ ID NO.2)；
amplifying an upstream primer and a downstream primer by using PCR to obtain an sgRNA in-vitro transcription template, transcribing the sgRNA in vitro to synthesize the sgRNA, and storing the sgRNA at a temperature of between 60 ℃ below zero and 95 ℃;
③ 0.5-5 mul of Cas9mRNA and sgRNA are evenly mixed to form a mixed solution, and the mixed solution is stored on ice;
dividing the fertilized eggs of the zebra fish in the step 1) into an injection group and a control group, and microinjecting 1nl of mixed solution to the interface of yolk and cells of the fertilized egg cells of 1-2 generations in the injection group;
culturing the zebra fish fertilized egg cells of the injection group and the control group in an incubator at 25-31 ℃, and replacing the culture solution at night on the day of injection;
3) obtaining stable genetic zebra fish homozygous lines:
culturing embryos which are split and developed by the injected group oosperms to adult individuals, carrying out tail shearing identification on the adult individuals, and screening a mutant F0 generation;
② crossing adult zebra fish F0 with wild zebra fish to obtain heterozygous F1 generation;
and thirdly, selfing the adult heterozygous zebra fish F1, and screening to obtain the homozygous zebra fish F2.
4) Construction of obese type II diabetes zebra fish model
Firstly, normally feeding the homozygous zebra fish F2 screened in the step 3) for 3 months by using the fairy shrimp as a feed;
secondly, after the cooked yolk added with 10% cholesterol is used as feed to feed for 12-18 days, the blood sugar of the zebra fish of the control group and the zebra fish cultured by the injection group is detected, the blood sugar content of the zebra fish of the injection group after 1.5 hours and 8 hours after the meal is more than 1.5 times of the blood sugar content of the zebra fish of the control group, and the establishment success of the obese type II diabetes zebra fish model can be judged.
2. The method for constructing the obese type II diabetic zebra fish model according to claim 1, wherein the ratio of the male and female wild zebra fish in the step 1) is 1:2 or 1: 1.
3. The method for constructing the obese type II diabetic zebrafish model according to claim 1, wherein the preparation method of the culture solution in the step 2) comprises the following steps: 200ml of KC solution with the concentration of 0.5 wt%, 200ml of CaCl2 solution with the concentration of 1 wt% and 200ml of NaHCO3 solution with the concentration of 0.25 wt% are mixed, 70g of solid NaCl and a proper amount of ddH2O are added to the mixture to be dissolved, the volume is kept to 20L, and then 26ml of methylene blue solution is added.
4. The method for constructing the obese type II diabetic zebrafish model according to claim 1, wherein the concentration of Cas9mRNA before mixing in the third step of step 2) is 600ng/μ l, and the concentration of sgRNA is 300ng/μ l.
5. The application of the obese type II diabetic zebra fish model obtained by the construction method according to any one of claims 1 to 4, wherein the obese type II diabetic zebra fish model is applied to the research of the pathological mechanism of diabetes or the screening and evaluation of medicines for treating type II diabetes.
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