CN114934074A - Construction method of ApoC3 gene knockout hamster model - Google Patents
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Abstract
The invention belongs to the technical field of construction of disease animal models, and particularly relates to a construction method of an ApoC3 gene knockout hamster model, which comprises three steps of collecting and culturing hamster zygotes, then microinjecting, and finally retransfusion of the zygotes.
Description
Technical Field
The invention belongs to the technical field of construction of disease animal models, and particularly relates to a construction method of an ApoC3 gene knockout hamster model.
Background
Apolipoprotein C3(ApoC3) is a small secreted glycoprotein with a molecular weight of 8.8kDa, synthesized predominantly by the liver and small intestine, and predominantly localized to circulating triglyceride-rich lipoproteins (TRLs), such as Chylomicrons (CM) and Very Low Density Lipoproteins (VLDL). The key function of ApoC3 is to inhibit lipoprotein lipase (LPL) -mediated hydrolysis of TG on TRL, impairing the clearance of TRL residues by lipoprotein receptors in the liver. At the same time, ApoC3 also stimulates VLDL secretion by the liver, disrupting the normal function of High Density Lipoprotein (HDL). Therefore, apolipoprotein C3 is an important regulator of plasma triglyceride, and the relationship between apolipoprotein C3 and hypertriglyceridemia has been well established.
Therefore, in order to better understand the relationship between ApoC3 and lipid metabolism and atherosclerosis, a construction method of an ApoC3 gene knockout hamster model is provided.
Disclosure of Invention
In order to make up for the defects of the prior art and better understand the relationship between ApoC3 and lipid metabolism and atherosclerosis, the invention provides a method for constructing an ApoC3 gene knockout hamster model.
The technical scheme adopted by the invention for solving the technical problem is as follows: a method for constructing an ApoC3 gene knockout hamster model comprises the following steps:
s100: collecting and culturing hamster zygotes;
s200: microinjection;
s300: and (5) returning the fertilized eggs.
Preferably, the step S100 includes the following sub-steps:
s101: determining the estrus cycle of the female mouse;
s102: superovulation of the egg mouse;
s103: preparing a pseudopregnant mouse;
s104: taking eggs and culturing fertilized eggs.
Preferably, in the step S102, female mice with the body weight of 130-150g and the age of 91-150 days are preferred when the egg-bearing mice are supervolved.
Preferably, in the step S102, transgene preparation is performed for 5 days after the superovulation of the oogenesis mice.
Preferably, in the substep S103, a male hamster with age of 6-8 weeks is selected for vasoligation.
Preferably, in the substep S104, the following substeps are adopted:
s1041: firstly, 70 mu L/drop of HECM-10 culture medium is covered with paraffin oil, the mixture is placed in an incubator and used for culturing fertilized eggs, and M2 and the HECM-10 culture medium are preheated at 37 ℃ and used for taking eggs;
s1042: making a vaginal smear on a female mouse, and taking the female mouse with sperm observed in the vaginal smear as an egg-supplying mouse;
s1043: preheating a culture medium for one hour, anesthetizing an egg-feeding mouse by using 3% sodium pentobarbital (3.5 mu L/g), opening the abdominal cavity, taking out an oviduct, placing the oviduct in the preheated M2 culture medium, taking out an egg mass from the ampulla of the oviduct under a body type microscope, repeatedly blowing the egg mass to obtain a single scattered fertilized egg, transferring the fertilized egg into an HECM-10 culture medium, placing the fertilized egg in an incubator for culture, and culturing the fertilized egg after micromanipulation in vitro for 1-2 hours.
Preferably, the S104 substep is performed for the fifth day of transgene preparation.
Preferably, Cas9mRNA and gRNA are selected for microinjection in the S200 step.
Preferably, the step S300 of reinfusing fertilized eggs means that after a pseudopregnant female mouse is anesthetized, the oviducts are pulled out through an opening on the back, and reinfusing umbrella orifices of the oviducts on both sides is performed, wherein 15 to 20 microinjected fertilized eggs are reinfused in each oviduct, and the gestation period of the hamsters is 17 days.
The invention has the following beneficial effects:
the ApoC 9 technology is applied to syrian golden hamster by constructing an ApoC3 knockout animal model, which is a small rodent model similar to human beings in the aspect of lipid metabolism characteristics, and the ApoC3 knockout (ApoC3-/-) hamster generated by CRISPR/Cas9 can better understand the relationship between ApoC3 and lipid metabolism and atherosclerosis, so that the ApoC 353532 knockout animal model becomes a gene modification animal model and is an important tool for researching gene function and the relationship between the ApoC and atherosclerosis.
Drawings
The invention will be further explained with reference to the drawings.
Fig. 1 is a design of a second exon-specific sgRNA of hamster ApoC 3;
FIG. 2 shows the result of PCR electrophoresis genotype identification of hamster DNA knocked out by ApoC3 gene;
FIG. 3 shows the results of RT-PCT for hamster liver with ApoC3 gene knockout;
FIG. 4 is SDS-PAGE of hamster plasma with ApoC3 gene knock-out;
FIG. 5 shows the ApoC3 knockout hamster plasma TG \ TC \ HDL-C;
FIG. 6 is an ApoC3 gene knockout homozygous hamster Oral Fat Load;
FIG. 7 shows the secretion of VLDL, homozygous for ApoC3 gene knock-out;
FIGS. 8-9 are ApoC3 knock-out homozygous hamster lipoprotein disk electrophoreses;
FIG. 10 is a ApoC3 knock-out homozygous hamster plasma western blot;
FIGS. 11-13 are ApoC3 gene knock-out homozygote hamster plasma TG \ TC \ HDL-C following HCHF feeding;
FIG. 14 is a disc electrophoresis of lipoprotein apoC3 knockout homozygous hamsters after feeding HCHF;
FIG. 15 is a ApoC3 gene knock-out homozygous hamster plasma western blot after HCHF feeding;
FIGS. 16-17 show the atherosclerosis profile of the fed HCHF.
Detailed Description
The present invention will be further described with reference to the following detailed description so that the technical means, the creation features, the achievement purposes and the effects of the present invention can be easily understood.
As shown in fig. 1 to 17, the method for constructing ApoC3 knockout hamster model according to the present invention comprises the following steps:
s100: collection and culture of hamster zygotes comprising the following substeps:
s101: determining the estrus cycle of the female mouse:
female hamsters typically mature sexually at 8 weeks of age, after which a stable estrus cycle occurs. The estrus cycle is typically 4 days, with the first day of the estrus cycle being estrus. On the next day of the estrus cycle, white viscous secretions appear at the vaginal opening, and the secretions can be used as a mark for judging the time course of the estrus cycle.
S102: superovulation of egg-feeding mice:
under appropriate conditions, estrus in female hamsters can occur throughout the year, and superovulation can be performed all the year round. The weight of the hamster is related to the superovulation effect, and the superovulation of female mice with the weight of 130-150g interval is most suitable. From the age, the superovulation effect is the best for 91-150 days old female mice. It is important that the time of superovulation coincides with the estrus cycle.
Transgene preparation day one: female mice on day two of the estrus cycle (white, sticky and exudative vaginal opening) were selected at 12 pm: 00-13: 00 intraperitoneal injection of PMSG20 IU;
fifth day of transgene preparation: before taking eggs, female mice are made into vaginal smears, and if sperm is found, the mating is proved to be successful, and the female mice can be used as egg-supplying mice.
S103: preparation of pseudopregnant mice: vasectomization was performed on 6-8 week old male hamsters. Fasting was performed for 6h before surgery, and anesthesia was performed with 3% sodium pentobarbital (3.5. mu.L/g). After fixation, the surgical area was shaved, disinfected, and a 0.5cm long incision was made in the lower abdomen on both sides, followed by sequential cutting of the skin and subcutaneous tissues. The tissue was bluntly dissected to form an incision about 1cm long, the epididymal fat was clamped to pull the vas deferens, and the vas deferens was severed by a cauterized forceps, and the 1cm length was removed. The vas deferens is then returned to the abdominal cavity. And (5) sewing the wounds in layers. The operation on the opposite side vas deferens is the same as the above operation. The wound is healed around the periphery after the operation, the male rat can be successfully mated with the female rat, and the success of the operation is proved when no sperm is detected in the semen, so that the male rat can be used for mating the surrogate mother rat. Transgene preparation day four 20: 00-22: 00, normal female mice on the first day of the estrus cycle are caged with successfully ligated male mice and mated. If there are more than 10 mating events, the induction of pseudopregnancy in the female mouse is considered successful, and such female mouse can be considered as a surrogate mouse.
S104: taking eggs and culturing fertilized eggs:
on the fifth day of transgene production, the following sub-steps were performed:
s1041: first, 70. mu.L/drop of HECM-10 medium was overlaid with paraffin oil and placed in an incubator for the cultivation of fertilized eggs. M2 and HECM-10 medium were pre-warmed at 37 ℃ for ova aspiration.
S1042: the female mice are used as vaginal smears, and the female mice with sperms observed in the vaginal smears are used as egg-supplying mice.
S1043: after warming up the medium for one hour, the egg-donor mice were anesthetized with 3% sodium pentobarbital (3.5 μ L/g), the abdominal cavity was opened, and the oviducts were removed and placed in a warmed-up M2 medium. Under a body microscope, the egg masses are taken out from the ampulla of the fallopian tube, and are repeatedly blown to obtain single scattered fertilized eggs. Transferring the fertilized eggs into a HECM-10 culture medium, and placing the fertilized eggs into an incubator for culture.
S200: microinjection;
fertilized eggs are transferred to M2 culture medium, and the drops of the culture medium are covered with paraffin oil for microinjection. Cas9mRNA was mixed with gRNA with the gene target sequence of interest, diluted to concentrations: cas9mRNA, 100 ng/. mu.L; gRNA, 25 ng/. mu.L. Then injected into the fertilized cytoplasm, and the liquid enters the cytoplasm under the microscope to indicate that the injection is successful.
Culturing the fertilized eggs after the micromanipulation in vitro for 1-2 hours.
S300: returning fertilized eggs;
after the pseudopregnant female mouse is anesthetized, the oviduct is pulled out from the opening of the back, and the two-side oviduct umbrella mouth reinfusion is carried out. 15-20 fertilized eggs after microinjection are reinfused by each oviduct. The gestational period of the hamsters was 17 days.
The gene modified animal model is an important tool for researching gene function and relation between the gene function and diseases, and has significance for constructing ApoC3 knockout animal model:
apolipoprotein C3(ApoC3) is a small secreted glycoprotein with a molecular weight of 8.8kDa, synthesized predominantly by the liver and small intestine, and predominantly localized to circulating triglyceride-rich lipoproteins (TRLs), such as Chylomicrons (CM) and Very Low Density Lipoproteins (VLDL). The key function of ApoC3 is to inhibit lipoprotein lipase (LPL) -mediated hydrolysis of TG on TRL, impairing the clearance of TRL residues by lipoprotein receptors in the liver. At the same time, ApoC3 also stimulates VLDL secretion by the liver, disrupting the normal function of High Density Lipoprotein (HDL). Therefore, apolipoprotein C3 is an important regulator of plasma triglycerides, and the relationship between apolipoprotein C3 and hypertriglyceridemia has been well established.
Therefore, to better understand the relationship between ApoC3 and lipid metabolism and atherosclerosis, the CRISPR/Cas9 technique was applied to syrian golden hamster, a small rodent model that is similar to humans in terms of lipid metabolism characteristics. An ApoC3 knockout (ApoC3-/-) hamster is generated by CRISPR/Cas 9.
The foregoing shows and describes the general principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. A method for constructing an ApoC3 gene knockout hamster model, which is characterized by comprising the following steps: the method comprises the following steps:
s100: collecting and culturing hamster zygotes;
s200: microinjection;
s300: and (5) returning the fertilized eggs.
2. The method of claim 1, wherein the ApoC3 knockout hamster model is constructed by: the step S100 includes the following substeps:
s101: determining the estrus cycle of the female mouse;
s102: superovulation of the egg mouse;
s103: preparing a pseudopregnant mouse;
s104: taking eggs and culturing fertilized eggs.
3. The method of claim 2, wherein the ApoC3 gene knockout hamster model is constructed by:
in the step S102, female rats with the weight of 130-150g and the age of 91-150 days are preferably selected when the female rats are superfertilized.
4. The method of claim 3, wherein the ApoC3 knockout hamster model is constructed by:
in the step S102, after the ovulatory mice are superovulation, transgenic preparation is carried out for 5 days.
5. The method of claim 2, wherein the ApoC3 knockout hamster model is constructed by: in the substep S103, a male hamster of 6-8 weeks old is selected for vasoligation.
6. The method of claim 2, wherein the ApoC3 knockout hamster model is constructed by: in the substep S104, the following steps are adopted:
s1041: firstly, 70 mu L/drop of HECM-10 culture medium is covered with paraffin oil, the mixture is placed in an incubator and used for culturing fertilized eggs, and M2 and the HECM-10 culture medium are preheated at 37 ℃ and used for taking eggs;
s1042: making a vaginal smear on a female mouse, and taking the female mouse with sperm observed in the vaginal smear as an egg-supplying mouse;
s1043: preheating a culture medium for one hour, anesthetizing an egg-feeding mouse by using 3% sodium pentobarbital (3.5 mu L/g), opening the abdominal cavity, taking out an oviduct, placing the oviduct in the preheated M2 culture medium, taking out an egg mass from the ampulla of the oviduct under a body type microscope, repeatedly blowing the egg mass to obtain a single scattered fertilized egg, transferring the fertilized egg into an HECM-10 culture medium, placing the fertilized egg in an incubator for culture, and culturing the fertilized egg after micromanipulation in vitro for 1-2 hours.
7. The method of claim 6, wherein the ApoC3 gene knockout hamster model is constructed by: the S104 substep is performed for the fifth day of transgene preparation.
8. The method of claim 1, wherein the ApoC3 gene knockout hamster model is constructed by: cas9mRNA and gRNA were selected for microinjection in the step S200.
9. The method of claim 1, wherein the ApoC3 knockout hamster model is constructed by: and the step S300, fertilized egg reinfusion, namely, anesthetizing a pseudopregnant female mouse, opening the back, pulling out the oviduct, and reinfusing the umbrella opening of the oviduct on two sides, reinfusing 15-20 fertilized eggs subjected to microinjection to each oviduct, wherein the pregnancy period of the hamster is 17 days.
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| CN103547271A (en) * | 2011-04-27 | 2014-01-29 | Isis制药公司 | Modulation of apolipoprotein CIII (APOCIII) expression |
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| YIWEN ZHA等: "CRISPR/Cas9-mediated knockout of APOC3", 《LIPIDS IN HEALTH AND DISEASE》 * |
| 卢瑶瑶: "运用CRISPR/Cas9基因编辑技术制备载脂蛋白C3基因敲除兔模型的相关研究", 《中国优秀硕士学位论文全文数据库 (基础科学辑)》 * |
| 魏群: "《生物工程技术实验指导》", 30 December 2002, 高等教育出版社 * |
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