CN112335581A - Fertilized egg for gene knockout and overexpression research of channel catfish and preparation method thereof - Google Patents
Fertilized egg for gene knockout and overexpression research of channel catfish and preparation method thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention discloses a fertilized egg for knocking out and over-expressing research of Ictalurus punctatus gene and a preparation method thereof, the method comprises parent fish cultivation, intensified ripening cultivation, parent fish selection, spawning induction of female parent fish, sperm solution preparation, ovum collection and artificial insemination, and the hatchability of the fertilized egg prepared by the method exceeds 80 percent after microinjection. The invention also provides an oxytocic for promoting ovulation of female parent fish of channel catfish and a dosage thereof, and the oviocic can promote the female parent fish to lay eggs and realize artificial egg taking after being injected. The fertilized eggs of the ictalurus punctatus prepared by the method can ensure the synchronous development of the fertilized eggs and meet the research requirements of gene knockout, overexpression and the like.
Description
Technical Field
The invention relates to methods for induced spawning and artificial insemination of fishes, in particular to a method for preparing fertilized eggs for gene knockout and overexpression research of channel catfish.
Background
Microinjection is a common experimental technique for molecular and cell biology research, which delivers minute quantities of substances (such as DNA, RNA, proteins, and other macromolecules) into cells or embryos, primarily through capillary glass tubes. Microinjection techniques have been used by researchers for gene knock-out, overexpression, and modification. In the research of gene knockout and overexpression of various organisms, microinjection is generally required to be carried out at the fertilized egg 1 cell stage, so that the obtainment of synchronous fertilized eggs is very critical.
The ictalurus punctatus roe is a sticky roe, tens of thousands of roes are adhered to form a thick egg block, and the embryo development after fertilization is not synchronous, so that the microinjection experiment is not facilitated. Although a plurality of literatures at home and abroad have researches on artificial spawning induction technology of the ictalurus punctatus, all of the literatures are used for improving the natural spawning rate after induction of parent fishes, and artificial egg extrusion and artificial insemination cannot be realized after injection of an oxytocic and a dosage in the literatures. The invention aims to provide a fertilized egg preparation method for gene knockout and overexpression research of channel catfish, which can ensure that the fertilized eggs are maintained to be fertilized synchronously and arranged in a monolayer manner. The invention also provides an oxytocic and an injection dosage for promoting ovulation of female parent fish of channel catfish, and artificial egg extrusion can be realized according to the components, the dosage and the injection method of the oxytocic provided by the invention so as to complete artificial insemination. The fertilized eggs of the Ictalurus punctatus prepared by the method can be ensured to be in the same development stage, and the researches on gene editing, overexpression and the like are met.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a fertilized egg for gene knockout and overexpression research of channel catfish and a preparation method thereof.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for preparing fertilized eggs for gene knockout and overexpression research of channel catfish, which comprises the following steps of (1) parent fish breeding: from G2Breeding the offspring seeds of the generation family to 4 years old to obtain parent fish; (2) strengthening and accelerating maturation culture: strengthening and accelerating maturity of 4-year-old parent fish in 3 months before breeding, and supplementing lecithin and compound vitamins during feeding; (3) parent (R)Selecting fishes: selecting healthy female and male parent fishes with clear and distinguishable second sexual characteristics, and placing the selected female fishes in a self-made spawning bag; (4) hasten parturition of female parent fish: the female parent fish is injected with oxytocin to promote ovulation; (5) preparing a sperm solution: after male parent fish is anesthetized, dissecting, and collecting spermary tissue to prepare a sperm solution; (6) collecting the ovum: after the female parent fish is anesthetized, a layer of plant shortening is coated around a cloaca and a ventral fin, the abdomen of the female parent fish is slightly pressed, and eggs are collected in a culture dish coated with the plant shortening in advance; (7) artificial insemination: adding a proper amount of sperm solution to the surface of the ovum, slightly stirring by using a medicine spoon coated with shortening, adding more fresh water to immerse the ovum, and keeping the fertilized ovum firm for 10-15 min to obtain the fertilized ovum which can be used for gene knockout and overexpression research of channel catfish.
Preferably, in step (1), G is2The generation family offspring seed is formed by one or more of Texas group, Acken group, Mississippi group, Acken group and Acken group through generation-by-generation directional construction and cultivation.
Preferably, in step (4), the oxytocin comprises chorionic gonadotropin and luteinizing hormone releasing hormone A2And domperidone (one or more of them).
Preferably, in the step (4), the female parent fish is induced to spawn by using a three-needle method, wherein the injection dosage is calculated by the weight of the parent fish, and the first needle: luteinizing hormone releasing hormone A250 ug/kg; a second needle: luteinizing hormone releasing hormone A26ug/kg, domperidone 3mg/kg, chorionic gonadotropin 500 IU/kg; and a third step of: luteinizing hormone releasing hormone A23ug/kg, 1.5mg/kg domperidone, 250IU/kg chorionic gonadotropin, and the injection time interval is 12 h.
Preferably, in the step (4), the female parent fish is injected with oxytocin, and after the oxytocin is injected, the female parent fish is placed back to the water tank, and the effect time is 14-20 hours under the condition that the water temperature is 20-25 ℃.
Preferably, in the step (3), the self-made egg laying bag has a specification of 100cm × 30cm, a mesh size of 3mm and is made of a soft nylon mesh.
Preferably, in the step (5) and the step (6), the anesthetic is 0.1% of 3-ethyl carbamate methanesulfonate.
Preferably, in step (7), the ovum obtained after fresh water immersion is capable of synchronous fertilization and development.
As another aspect of the present invention, the present invention provides a fertilized egg for knocking out and overexpressing ietalurus punetaus gene, comprising: after microinjection is carried out on the fertilized eggs, the hatchability of the fertilized eggs exceeds 80 percent.
Preferably, the microinjection must be completed within 90 min.
Compared with the prior art, the invention has the beneficial effects that:
(1) the components, the dosage and the injection method of the oxytocic for hastening the spawning of the female parent fish are provided, the female parent after the oxytocic treatment can adopt an artificial egg squeezing mode to collect eggs, and the artificial insemination is completed;
(2) the ietalurus punetaus fertilized eggs prepared by the artificial insemination mode have a synchronous embryo development process, and researchers can accurately master the embryo development period;
(3) the fertilized egg of the ictalurus punctatus prepared according to the specification can be used for researches such as gene editing, overexpression and the like, and the fertilized egg after microinjection has high hatchability.
Detailed Description
The present invention will be further described below.
Example 1
A fertilized egg preparation method for gene knockout and overexpression research of channel catfish comprises the following steps:
(1) parent fish rearing
The channel catfish parent fish used in the experiment is from the experimental base raised by the fresh water aquatic product research institute of Jiangsu province. Breeding base populations were established in 1997-2004 from the United states after introduction of the Texas (1997) population, the Ackerel (1999) population, the Mississippi (2001) population, the Ackerel (2003) population, and the Ackerel (2004) population 405, and G was performed using the base populations0And constructing a generation family. 6-month profit in 2013By G0Ancestry construction G1Ancestry, 2017 use G1Generation family parent construction G2And (5) family generation. The parent used in this experiment consists of G2The method is characterized in that the method is formed by breeding of family offspring seeds, in order to reduce the influence of the environment on the growth of the channel catfish, the family breeding is carried out according to the same standard, namely the uniform water changing rate (water is changed every 10 days, the water changing amount is 1/3 of a water body), the feeding amount (1 time of feeding in the morning and at night every day, the feeding amount is 1% -3% of the weight of the fish, the feeding amount is adjusted every 10 days), and the breeding density (20 tails/m)2) And the oxygen filling amount is 5 mg/L-8 mg/L. When the average day age of each family is 150d, PIT electronic mark injection is carried out to distinguish different families and identities. Then, each family is mixed and cultivated, and the cultivation density is 1000 tails/mu. Feeding with artificial expanded pellet feed, wherein the content of crude protein in the artificial expanded pellet feed is more than or equal to 32 percent, the content of crude fat is more than or equal to 6 percent, the content of crude fiber is less than or equal to 4 percent, and the ash content is less than or equal to 14 percent, feeding the artificial expanded pellet feed 2 times a day, the daily feeding amount is about 3 percent of the weight of the fish, the actual feeding amount is adjusted according to the conditions of weather, water temperature and the like, and the specifications of the fed feed including the particle size and the content of each component are adjusted according to the size of the fish along with the growth of the; meanwhile, daily management and water quality regulation and control of seedling culture are enhanced, and disease control work is well done. The parent fish can be ripened and propagated when being cultured to 4 years old.
(2) Intensified ripening cultivation of parent fish
The intensive ripening cultivation is carried out 3 months before the 4-year-old parent fishes are bred, and the feed sprayed with lecithin and the frozen fresh trash fish bait are fed every day. The iced fresh trash fish bait is fed for 1 time every morning, the daily feeding amount is 1-3% of the weight of the fish, and the compound vitamin is added according to the weight of the iced fresh bait according to 2 g/kg. The puffed feed containing lecithin is fed once every afternoon, the daily feeding amount is 1% -3% of the weight of fish, the crude protein content of the feed is more than or equal to 40%, the crude fat is more than or equal to 6%, the crude fiber is less than or equal to 4%, and the ash content is less than or equal to 14%. The actual feeding amount is properly adjusted according to the conditions of weather, water temperature and the like so as to avoid feeding excessive baits. The bait for feeding the iced fresh trash fish is extremely easy to damage water, in order to ensure that the parent fish has good water quality conditions in the maturing and strengthening cultivation link, the water needs to be changed periodically, the water changing amount is about 1/3 of the total water amount of the pond, and meanwhile, related bottom-changing biological agents are sprayed to improve the water quality.
(3) Selection of parent fish
And selecting healthy channel catfish parent fish from each family, and ensuring that no relationship exists between the selected male and female parent fish, and the male and female parent fish should have a clear and distinguishable second sex character. The selection of parent fish was performed according to the following criteria: the male fish body is dark, and the head is wider than other parts of the body; female fish have a soft and full abdomen, and have a narrower head than other parts of the body. The weight of the male fish is 3-4 kg, and the weight of the female fish is 2-3 kg. The action of picking up the parent fishes is too large to avoid causing stress to the parent fishes, and feeding is stopped for 2-3 days before the parent fishes are picked up.
In the experiment, 3 tails of male parent fishes and 18 tails of female parent fishes of different families are selected. The female parent fish is placed in a self-made spawning bag (100 cm multiplied by 30cm, the mesh size is 3mm, and the material is a soft nylon net), the spawning bag filled with the female parent fish is placed in a water pool (3m multiplied by 1m multiplied by 0.5m) filled with water, and the subsequent injection of an oxytocic and ovulation observation are facilitated. The spawning bag mouth is fixed at the edge of the water pool, and the water pool is kept in a micro-flowing state and has sufficient oxygen. The male parent fish can be placed in the water pool without being placed in the spawning bag.
(4) Hasten parturition of female parent fish
Before female parent fish induced spawning, the weight of each fish was weighed and recorded. The oxytocin is prepared from oxytocin and 0.9 wt% NaCl solution, and the oxytocin includes chorionic gonadotropin (HCG) and luteinizing hormone releasing hormone A2(LHRH-A2) Domperidone (Dom) (Ningbo second hormone plant). The oxytocic is prepared according to the body of the parent fish before injection, and each fish is prepared in 1 tube. An oxytocic is injected into the base part of the pectoral fin, the injection depth is 0.5-0.6 cm, the base part of the pectoral fin is wiped with 0.9% NaCl (Shanghai national drug group chemical agent Co., Ltd.) solution before injection, and a 75% alcohol solution is used for wiping the wound after injection. The artificial induced spawning adopts a three-needle method, the time interval of each injection is 12h, two dosage groups are set, and 9 parent fishes are tested in each dosage group. The first dose group was: first needle, LHRH-A250 ug/kg; second needle, LHRH-A26ug/kg, Dom 3mg/kg, HCG 500 IU/kg; third, each oxytocin dose is half of the second (i.e. LHRH-A)23ug/kg, Dom 1.5mg/kg, HCG 250 IU/kg). The second dose group was: first needle, LHRH-A2100 ug/kg; second needle, LHRH-A212ug/kg, Dom 6 mg/kg, HCG 1000 IU/kg; third, each oxytocin dose is half of the second (i.e. LHRH-A)26ug/kg, Dom 3mg/kg, HCG 500 IU/kg). The female parent fish can be observed to have more prominent and red and swollen genital pores 6-8 hours after the second injection of the oxytocic. After the oxytocic is injected, the spawning bag is put back to a water pool, sufficient oxygen (dissolved oxygen of about 6 mg/L) and good water quality are maintained to ensure that high-quality ova are produced, the effect time is 14-20 h under the condition that the water temperature is 20-25 ℃, and the injection amount, the effect time and the survival condition of each female parent fish are shown in table 1.
(5) Collection of sperm
Sperm solutions are prepared prior to the pre-spawning of the female fish. Male parent fish were anesthetized with 0.1% ethyl-3-aminobenzoate methanesulfonate (MS-222). After male parent fish is anesthetized, the spermary tissue is dissected and collected, the weight is weighed, 50mL of 0.9% NaCl solution is measured to clean blood adhered to the surface, and the good spermary tissue is fibrous and milky. The spermary tissue was transferred to a silk net (area 100 cm) with forceps2And the mesh number is 100), wrapping the spermary tissue by folding a silk net, continuously extruding the wrapped spermary tissue at a 50mL centrifugal nozzle to extract sperms, and simultaneously washing semen adhered to the silk net into a centrifugal tube by using 0.9% NaCl solution, wherein the final volume mass ratio of the NaCl solution to the spermary tissue is 10 mL/g. The sperm solution is stored at 4 ℃ and used within 24 h.
(6) Collection of ova
After the third injection of the oxytocic, checking the ovulation condition every 3h, and starting manual egg squeezing if more than 10 eggs attached to the egg laying bag are found. The female fish was anesthetized by placing it in MS-222(Merck, Germany) at a concentration of 0.1%. Before egg extrusion, a clean towel is used for wiping female parent fish, and a layer of plant shortening (Nutiva company, USA) is coated around the cloaca and the ventral fin so as to prevent the eggs from adhering to the cloaca or the ventral fin during egg extrusion. The abdomen of the female parent fish was gently pressed and the eggs were collected in a dish (150mm) previously coated with vegetable shortening. The good ovum can slide freely, the color is golden yellow, no blood clot exists, the diameter of the ovum is 3-4 mm, and the ovum is prevented from contacting water before fertilization because the water stimulates the closing of the ovum membrane hole. In the first dosage group of the experiment, 9 female parent fishes are induced to spawn together, wherein 8 female parent fishes can successfully extrude eggs within 12-20 hours after the third injection, the specific spawning time is shown in table 1, and the combination is indicated as an effective dosage; more than half of the parent fish in the second dose group died, indicating that the dose of the combination is not suitable for induction of spawning by female parent fish.
TABLE 1 female parent fish oxytocic injection and ovulation status recording sheet
Note: + indicates ovulation and-indicates non-ovulation.
(7) Artificial insemination
An appropriate amount of sperm solution was added to the surface of the eggs, gently stirred using a spatula coated with shortening, approximately 1mL of sperm solution per 100 eggs. After a proper amount of clear water is added, the shaking is carried out for 30s, and the activity of ova and sperms can be stimulated in a fresh water environment. Lightly stirring the fertilized eggs by using a medicine spoon coated with shortening oil to keep the fertilized eggs in a monolayer arrangement state, adding a proper amount of fresh water to immerse the fertilized eggs, and keeping the fertilized eggs firm for 10-15 min. The fertilized egg of the Ictalurus punctatus obtained by the method can be used for microinjection experiments.
(8) Microinjection
Coating a thin layer of plant shortening on a 10cm sterile culture dish, transferring 50-100 fertilized eggs into the culture dish, keeping the fertilized eggs in a monolayer arrangement state, and sucking a pre-prepared Holtfreter solution (100%) to immerse the fertilized eggs. The culture dish containing the fertilized egg is placed under a microscope, one hand holds the culture dish, and the other hand lightly controls the operating rod of the microinjection instrument to ensure that the operating rod pierces the egg membrane and the yolk in a smooth motion. The capillary tip should be as close to the blastoderm as possible before the infusion is delivered. The pedal is depressed to deliver the injectate to the yolk adjacent the animal pole. The injection dosage is different according to different research purposes, in the gene editing research, 30-50 nL of mixed solution of gRNA and Cas9 can be injected, and the injection amounts of the gRNA and Cas9 protein are required to be optimized for a target gene so as to obtain the optimal mutation rate. Fertilized eggs which have completed microinjection are placed back into the Holtfreter solution (100%) and incubated for 6-7d continuously. The solution is replaced every day, and the disinfection work is done, and 100ppm of the pyrimethanil solution can be used for disinfection.
It should be noted that the microinjection experiment must be completed within 90min, since the fertilized egg is at the 1-cell stage. Unfertilized eggs are covered with a preservative film to prevent drying, and artificial insemination can be performed in a staggered manner, for example, one batch of eggs can be fertilized every 15-30 min to ensure that enough fertilized eggs in the 1-cell stage are used for microinjection experiments. After microinjection experiments of the fertilized eggs of the channel catfish obtained by the scheme are carried out, the hatchability is more than 80%, which shows that the fertilized eggs of the channel catfish prepared by the scheme can meet the microinjection experiments of gene knockout, overexpression and the like.
According to the invention, a directional family construction technology is adopted to prepare a family breeding group of the channel catfish, the intensive ripening cultivation of parent fish is started when the channel catfish is cultivated to 4 years old, and the egg yolk lecithin-sprayed feed and the iced fresh fish are fed three months before artificial insemination is carried out, so that the gonad development of the parent fish is further promoted; selecting male fish with dark body color and wider head than other parts of the body; selecting female fish with narrower head than other parts of body, plump and soft abdomen, and red and swollen genitals; placing the selected female parent fish in a self-made spawning bag, preparing an oxytocic and injecting to realize artificial spawning induction of the parent fish; after selected male parent fish is anesthetized, dissecting, collecting spermary, weighing and weighing to prepare a sperm solution, and storing the sperm solution at 4 ℃; after anaesthetizing the female parent fish after the induced spawning treatment, collecting ova in a manual egg squeezing mode; and adding a proper amount of prefabricated sperm solution to the surface of the collected Ictalurus punctatus ovum to prepare a fertilized ovum.
The breeding population used in the present invention is G1The breeding population of the channel catfish is bred by a directed family construction technology. When the seedlings of each family are cultivated to the average age of 150 days, electronic marker injection is carried out to distinguish different families and identities. And (3) carrying the electronic marker, and carrying out mixed culture on the seedlings in the pond, wherein the culture density is 1000 tails/mu. Feeding with artificial puffed pellet feed, wherein the crude protein content of the artificial puffed pellet feed is more than or equal to 32%, the crude fat is more than or equal to 6%, the crude fiber is less than or equal to 4%, the ash content is less than or equal to 14%, feeding the artificial puffed pellet feed for 2 times a day, and the daily feeding amount is about 3% of the weight of the fish.
In the invention, the parent fish subjected to intensive culture is 4-year old fish, the intensive ripening culture starts in March, the duration is 3 months, and the feed sprayed with lecithin and the frozen fresh trash fish bait are fed in the culture process. The iced fresh trash fish bait is fed for 1 time every morning, the daily feeding amount is 8-10% of the weight of the fish, and the compound vitamin is added according to the weight of the iced fresh bait according to 2 g/kg. The puffed feed containing lecithin is fed once every afternoon, the daily feeding amount is 10-15% of the weight of fish, the crude protein content of the feed is more than or equal to 40%, the crude fat is more than or equal to 6%, the crude fiber is less than or equal to 4%, and the ash content is less than or equal to 14%.
In the invention, the male and female parent fishes are required to be robust, free of injuries and diseases, good in fullness and strong in mobility, the male parent fishes are dark in color, and the heads of the male parent fishes are generally wider than other parts of the bodies; the head of a female parent fish is generally narrower than the body and has a soft and full abdomen with prominent redness and swelling of the genitals. The weight of the selected parent fish is 3-4 kg, and the weight of the female parent fish is 2-3 kg.
The artificial induced spawning is carried out by a three-needle method, and the injection time interval is 12 hours. A first needle: LHRH-A250 ug/kg; a second needle: LHRH-A26ug/kg, Dom 3mg/kg, HCG 500 IU/kg; the oxytocin dosage of the third needle is half of that of the second needle, and is calculated by the weight of the fish body. The oxytocin injection is carried out in a pectoral fin base injection mode, and the injection depth is 0.5-0.6 cm. The base of pectoral fin was wiped with 0.9% NaCl solution before injection, and 75% NaCl solution was used after injection% alcohol solution. After the oxytocic is injected, the water dissolved in the pool is sufficient, and the surrounding environment is quiet; the effect time is 14-20 h under the condition that the water temperature is 20-25 ℃.
In the invention, the anesthesia strategy of the female and male parent fishes is to immerse the fish bodies in water containing 0.1% MS-222 for about 30-60 s. After the collection of the testis tissue, the weight was measured, and the blood adhered to the surface was washed with 50mL of 0.9% NaCl solution. The collection of sperms is mainly realized by extruding spermary tissues, and the main process is as follows: placing testis tissue at size of 100cm2The wrapped silk was extruded through a 50mL centrifugal nozzle. The sperm solution is formed by diluting 0.9% NaCl solution, and the final volume-mass ratio of the NaCl solution to the spermary tissue is 10 mL/g.
Before artificial egg squeezing, a layer of plant shortening is coated around cloaca and ventral fin to prevent egg adhesion. The artificial egg squeezing is mainly to lightly squeeze the abdomen of a female parent fish to discharge eggs, and the eggs are collected in a culture dish coated with vegetable shortening in advance. The sperm solution is used in an amount of about 1mL of sperm solution per 100 eggs. And (3) after adding a proper amount of clear water, shaking for 30s, lightly stirring by using a spoon coated with shortening oil to keep the fertilized eggs in a single-layer arrangement state, and adding more fresh water to immerse the fertilized eggs to keep the fertilized eggs stiff for 10-15 min. The obtained fertilized egg of the ictalurus punctatus can be subjected to microinjection and is used for researches such as gene editing and overexpression.
The invention provides a fertilized egg preparation method for gene knockout and overexpression research of channel catfish, which comprises the following steps: parent fish family cultivation, parent fish intensive ripening cultivation, parent fish selection, female parent fish induced spawning, sperm collection, ovum collection and artificial insemination. The invention provides an oxytocic for promoting ovulation of female parent fish of channel catfish and a dosage thereof, which can realize artificial egg taking. The fertilized eggs of the Ictalurus punctatus prepared by the method can be ensured to be in the same development stage, and the experimental requirements of gene editing, over-expression and the like are met.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. A fertilized egg preparation method for gene knockout and overexpression research of channel catfish is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
(1) parent fish breeding: from G2Breeding the offspring seeds of the generation family to 4 years old to obtain parent fish;
(2) strengthening and accelerating maturity cultivation: intensive ripening cultivation is started 3 months before 4-year-old parent fish are bred, and lecithin and compound vitamins are supplemented during feeding;
(3) selecting parent fish: selecting healthy female and male parent fishes with clear and distinguishable second sexual characteristics, and placing the selected female fishes in a self-made spawning bag;
(4) hasten parturition of female parent fish: the female parent fish is injected with oxytocin to promote ovulation;
(5) preparing a sperm solution: after male parent fish is anesthetized, dissecting, and collecting spermary tissue to prepare a sperm solution;
(6) collecting the ovum: after the female parent fish is anesthetized, a layer of plant shortening is coated around a cloaca and a ventral fin, the abdomen of the female parent fish is slightly pressed, and eggs are collected in a culture dish coated with the plant shortening in advance;
(7) artificial insemination: adding a proper amount of sperm solution to the surface of the ovum, slightly stirring by using a medicine spoon coated with shortening, adding more fresh water to immerse the ovum, and keeping the fertilized ovum firm for 10-15 min to obtain the fertilized ovum which can be used for gene knockout and overexpression research of channel catfish.
2. The method for preparing a fertilized egg for knocking out and over-expressing Ictalurus punctatus gene according to claim 1, wherein the fertilized egg is prepared by the following steps: in the step (1), the G2The generation family offspring seed is formed by one or more of Texas group, Ackeran group, Missippi group, Ackeran group and Ackeran group through generation-by-generation directional construction and cultivation.
3. Such asThe method for preparing a fertilized egg for knocking out and overexpressing ietalurus punetaus gene according to claim 1, which comprises the steps of: in step (4), the oxytocin comprises chorionic gonadotropin and luteinizing hormone releasing hormone A2And domperidone (one or more of them).
4. The method for preparing a fertilized egg for knocking out and over-expressing Ictalurus punctatus gene according to claim 3, wherein the fertilized egg is prepared by the following steps: in the step (4), the induced spawning of the female parent fish is realized by adopting a three-needle method, wherein the injection dosage is calculated by the weight of the parent fish, and the first needle: luteinizing hormone releasing hormone A250 ug/kg; a second needle: luteinizing hormone releasing hormone A26ug/kg, domperidone 3mg/kg, chorionic gonadotropin 500 IU/kg; and a third step of: luteinizing hormone releasing hormone A23ug/kg, 1.5mg/kg domperidone, 250IU/kg chorionic gonadotropin, and the injection time interval is 12 h.
5. The method for preparing a fertilized egg for knocking out and over-expressing Ictalurus punctatus gene according to claim 4, wherein the fertilized egg is prepared by the following steps: and (4) injecting oxytocin into the female parent fish, putting the female parent fish into a water tank after the oxytocin is injected, and enabling the female parent fish to have an effect time of 14-20 hours at the water temperature of 20-25 ℃.
6. The method for preparing a fertilized egg for gene knockout and overexpression research of Ictalurus punctatus as claimed in any one of claims 1 to 5, wherein the fertilized egg comprises: in the step (3), the self-made egg laying bag has the specification of 100cm multiplied by 30cm, the mesh size is 3mm, and the material is soft nylon net.
7. The method for preparing a fertilized egg for gene knockout and overexpression research of Ictalurus punctatus as claimed in any one of claims 1 to 5, wherein the fertilized egg comprises: in the step (5) and the step (6), the anesthetic is 0.1% of ethyl 3-aminobenzoate methanesulfonate.
8. The method for preparing a fertilized egg for gene knockout and overexpression research of Ictalurus punctatus as claimed in any one of claims 1 to 5, wherein the fertilized egg comprises: in the step (7), the ovum obtained after the immersion in fresh water can be fertilized and developed synchronously.
9. A fertilized egg for gene knockout and overexpression research of channel catfish is characterized in that: after microinjection is carried out on the fertilized eggs, the hatchability of the fertilized eggs exceeds 80 percent.
10. The fertilized egg for knocking out and over-expressing genes of Ictalurus punctatus according to claim 9, wherein: the microinjection must be completed within 90min after artificial insemination.
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