Method for separating zebra fish juvenile fish heart

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for separating zebrafish juvenile fish hearts. According to the method for separating the heart of the juvenile zebrafish, the heart of the juvenile zebrafish is located on the outer side relative to visceral tissue, the heart is wrapped by a layer of pericardium, the pericardium can be decomposed by trypsin for a specific time to expose the heart, then suction is conducted through external force, and the heart is separated. The heart is separated from the juvenile fish under the action of pancreatin and external force. By means of the separation method, the integrity of the separated zebra juvenile fish heart can be kept, the separation method is rapid and efficient, and a large number of juvenile fish hearts can be separated and dissected at the same time.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to a method for isolating the heart of zebrafish larvae. Background Art

[0002] Zebrafish are vertebrates that are externally fertilized, externally developed, have transparent embryos, and multiple organs can be regenerated, and have gradually become an important model vertebrate for studying embryonic development and organogenesis. The heart is the first functional organ during embryonic development, pumping blood and supplying oxygen to the body to promote its development. Approximately 8.9‰ of live newborns each year have congenital heart development defects. Therefore, exploring the gene functions during heart development and studying genetic mutations through model animals have important clinical significance.

[0003] During embryonic development, the embryo finally develops into an individual with multiple organs and complete functions through various signaling pathways and gene function regulations. Among them, the morphogenesis of organs occupies an important position in basic scientific research. To study the morphogenesis of organs and avoid the influence of other tissues and organs, organ anatomy has become the primary step in studying organogenesis.

[0004] Adult zebrafish organ anatomy is relatively simple, especially the anatomy of heart tissue, and the zebrafish heart can regenerate. Therefore, zebrafish are an ideal model animal for studying heart development and regeneration. However, the morphogenesis of the zebrafish heart occurs 12 hours post fertilization (12hpf), at which time cardiac progenitor cells are expressed in the anterior lateral plate mesoderm. As embryonic development progresses, it develops into a heart tube at 24hpf, and then undergoes morphogenetic movements such as looping and chamber dilation, and finally develops into a functional heart at 72hpf. Therefore, studying the morphogenesis of the heart requires dissecting the heart of larvae.

[0005] In traditional techniques, the dissection of the heart of larvae is mainly carried out using manual dissection instruments. However, due to the small size of the larvae and the even smaller size of the heart, the operation is highly complex, time-consuming, and the integrity of the dissected heart is poor, with varying degrees of heart damage and more tissues outside the heart adhering to it. Summary of the Invention

[0006] Based on this, an embodiment of this application provides a method for isolating the heart of zebrafish larvae, which can successfully isolate the heart of zebrafish larvae in a non-physical manner.

[0007] On the one hand, the present application provides a method for separating the heart of zebrafish larvae, including: providing zebrafish larvae embryos, performing dechorionation treatment on the embryos; digesting the dechorionated embryos with trypsin; and pipetting the product of the digestion treatment to prepare the separated heart of zebrafish larvae.

[0008] The developmental stage of the zebrafish larvae embryos is 24 hpf to 72 hpf, and the total incubation duration is 15 s to 62 s.

[0009] In one embodiment, the developmental stage of the zebrafish larvae embryos is 24 hpf to 26 hpf, and the total incubation duration is 14 s to 16 s.

[0010] When the developmental stage of the zebrafish larvae embryos is 35 hpf to 37 hpf, the total incubation duration is 28 s to 32 s.

[0011] When the developmental stage of the zebrafish larvae embryos is 46 hpf to 50 hpf, the total incubation duration is 58 s to 62 s.

[0012] When the developmental stage of the zebrafish larvae embryos is 70 hpf to 74 hpf, the total incubation duration is 58 s to 62 s.

[0013] In one embodiment, the components of the trypsin include one or more of lipase, nuclease, polysaccharide, and protease.

[0014] In one embodiment, the trypsin is trypsin-EDTA.

[0015] In one embodiment, the concentration of the trypsin is 0.20 w / v% to 0.30 w / v%.

[0016] In one embodiment, after digesting the dechorionated embryos with trypsin, it further includes a step of terminating the digestion.

[0017] In one embodiment, terminating the digestion includes using fetal bovine serum with a concentration of 8 v / v% to 12 v / v%.

[0018] In one embodiment, the dechorionation treatment includes using one or both of physical treatment and chemical treatment.

[0019] In one embodiment, the physical treatment includes using a puncture tool; in one embodiment, the puncture tool includes a needle with a diameter of 0.4 mm to 0.5 mm.

[0020] In one embodiment, the chemical treatment includes using pronase E dechorionation solution.

[0021] In one embodiment, the concentration of pronase stripping solution is 0.8 mg / mL to 2 mg / mL.

[0022] In one embodiment, before preparing the separated zebrafish larvae hearts, there is also a step of washing with a washing solution.

[0023] In one embodiment, the washing solution includes Ringer's solution.

[0024] Details of one or more embodiments of the present application are set forth in the following description. Other features, objects, and advantages of the present application will become apparent from the specification and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more fully understand the present application and its beneficial effects, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 Degree of separation and integrity results of the heart after trypsin digestion at different time points at 24 hpf;

[0027] Figure 2 Degree of separation and integrity results of the heart after trypsin digestion at different time points at 36 hpf;

[0028] Figure 3 Degree of separation and integrity results of the heart after trypsin digestion at different time points at 48 hpf;

[0029] Figure 4 Degree of separation and integrity results of the heart after trypsin digestion at different time points at 72 hpf. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present application will be further described in detail below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] The term

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein have the following meanings:

[0034] As used herein, the selection scope of the terms “and / or”, “or / and”, “and / or” includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from “and / or”, “or / and”, “and / or” are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by “logical AND”, and also undoubtedly includes the technical solution connected by “logical OR”. For example, “A and / or B” includes three parallel solutions: A, B, and A + B. Another example, the technical solution of “A, and / or, B, and / or, C, and / or, D” includes any one of A, B, C, D (that is, the technical solution connected by “logical OR”), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (that is, the technical solution connected by “logical AND”).

[0035] In this application, when it comes to “multiple”, “multiple kinds”, “multiple times”, “multiple elements”, etc., without special limitation, it means greater than 2 or equal to 2 in quantity. For example, “one or more kinds” means one kind or greater than or equal to two kinds.

[0036] As used herein, “its combination”, “any combination thereof”, “any combination mode thereof”, etc. include all suitable combination modes of any two or any two or more of the listed items.

[0037] In this article, “suitable combination mode”, “suitable mode”, “any suitable mode”, etc., the “suitable” therein is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0038] In this application, “further”, “even further”, “especially”, etc. are used for descriptive purposes and represent differences in content, but should not be construed as limiting the protection scope of this application.

[0039] In this application, "optionally", "optional", and "option" mean "may or may not", that is, any one of two alternative options of "yes" or "no". If "optional" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0040] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0041] In this application, regarding numerical intervals (i.e., numerical ranges), without special instructions, the optional numerical values distributed within the above numerical intervals are considered continuous, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions of features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0042] The temperature parameter in this application, without special limitations, allows both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0043] In this application, both %(w / w) and wt% represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0044] All documents mentioned in this application are cited as references in this application, just as if each document is cited separately as a reference. Unless it conflicts with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited for all contents and all purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited together. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0045] The term "zebrafish larvae" refers to zebrafish larvae with a transparent body, which allows researchers to directly observe the development of their internal organs and the operation of the blood circulation system. Zebrafish develop rapidly from fertilized eggs to the larval stage. The embryo is active and has a beating heart 24 hours after fertilization, hatches into a larva in about 3 days, and the small zebrafish can move freely and search for food 7 days later. In addition, the body length of zebrafish larvae is only about 3 mm, and their internal organs are small but each has its own function.

[0046] The term "hpf", which stands for hour post fertilization, is the time unit after zebrafish fertilization and is commonly used to describe the stages of embryonic development.

[0047] On the one hand, the present application provides a method for isolating the heart of zebrafish larvae, including: providing zebrafish embryos, performing dechorionation treatment on the embryos; digesting the dechorionated embryos with trypsin; and pipetting the product of the digestion treatment to prepare the isolated heart of zebrafish larvae. The preparation process includes picking the isolated zebrafish heart under a microscope.

[0048] The developmental stage of zebrafish embryos is 24 hpf to 72 hpf, and the total incubation duration is 15 s to 62 s.

[0049] The present application provides a method for isolating the heart of zebrafish larvae. The heart of zebrafish larvae is located outside relative to the visceral tissue, and there is a layer of pericardium outside the heart. This layer of pericardium can be decomposed by trypsin at a specific time to expose the heart, and then suction is performed by external force. Under the action of trypsin and external force, the heart is separated from the larvae. Through the isolation method of the present application, the integrity of the isolated heart of zebrafish larvae can be maintained, and it is fast and efficient. It can also simultaneously isolate and dissect the hearts of a large number of larvae.

[0050] For example, the developmental stages of zebrafish larvae embryos are 24 hpf, 25 hpf, 26 hpf, 27 hpf, 28 hpf, 29 hpf, 30 hpf, 31 hpf, 32 hpf, 33 hpf, 34 hpf, 35 hpf, 36 hpf, 37 hpf, 38 hpf, 39 hpf, 40 hpf, 41 hpf, 42 hpf, 43 hpf, 44 hpf, 45 hpf, 46 hpf, 47 hpf, 48 hpf, 49 hpf, 50 hpf, 51 hpf, 52 hpf, 53 hpf, 54 hpf, 55 hpf, 56 hpf, 57 hpf, 58 hpf, 59 hpf, 60 hpf, 61 hpf, 62 hpf, 63 hpf, 64 hpf, 65 hpf, 66 hpf, 67 hpf, 68 hpf, 69 hpf, 70 hpf, 71 hpf or 72 hpf, as well as any value in between.

[0051] For example, the incubation duration is 15 s, 16 s, 17 s, 18 s, 19 s, 20 s, 21 s, 22 s, 23 s, 24 s, 25 s, 26 s, 27 s, 28 s, 29 s, 30 s, 31 s, 32 s, 33 s, 34 s, 35 s, 36 s, 37 s, 38 s, 39 s, 40 s, 41 s, 42 s, 43 s, 44 s, 45 s, 46 s, 47 s, 48 s, 49 s, 50 s, 51 s, 52 s, 53 s, 54 s, 55 s, 56 s, 57 s, 58 s, 59 s, 60 s, 61 s or 62 s, as well as any value in between.

[0052] In some embodiments, when the developmental stage of zebrafish larvae embryos is 24 hpf - 26 hpf, the protease digestion treatment duration is 14 s - 16 s. For example, at the 24 hpf developmental stage, the trypsin digestion treatment duration is 14 s - 16 s, preferably 15 s. Treating the heart tissue with this duration can keep it relatively intact and can also separate it well from the embryo body.

[0053] In some embodiments, when the developmental stage of zebrafish larvae embryos is 35 hpf - 37 hpf, the protease digestion treatment duration is 28 s - 32 s. For example, at the 36 hpf developmental stage, the trypsin digestion treatment duration is 28 s - 32 s, preferably 30 s. Treating the heart tissue with this duration can keep it relatively intact and can also separate it well from the embryo body.

[0054] In some of these embodiments, when the developmental stage of the zebrafish larval embryo is 46 hpf to 50 hpf, the duration of protease digestion treatment is 58 s to 62 s. For example, at the 48 hpf developmental stage, the duration of trypsin digestion treatment is 58 s to 62 s, preferably 60 s. Treating the heart tissue for this duration can keep it relatively intact and can also separate it well from the embryo body.

[0055] In some of these embodiments, when the developmental stage of the zebrafish larval embryo is 70 hpf to 74 hpf, the duration of protease digestion treatment is 58 s to 62 s. For example, at the 72 hpf developmental stage, the duration of trypsin digestion treatment is 58 s to 62 s, preferably 60 s. Treating the heart tissue for this duration can keep it relatively intact and can also separate it well from the embryo body.

[0056] In some of these embodiments, trypsin includes one or more of lipase, nuclease, polysaccharide, and protease.

[0057] The trypsin is Gibco Trypsin-EDTA (0.25%), containing phenol red.

[0058] Among them, Gibco Trypsin-EDTA is made from trypsin powder (an irradiated mixture of proteases from porcine pancreas). Trypsin-EDTA (0.25%), containing phenol red, is a product that has been tested for swine fever virus and mycoplasma. It contains 2.5 g / L of trypsin (1:250) and 0.38 g / L of EDTA4Na in Hanks' Balanced Salt Solution, without CaCl2, MgCl 2。

[0059] In some of these embodiments, the concentration of trypsin is 0.20 w / v% to 0.30 w / v%. For example, the trypsin concentration is 0.2 w / v%, 0.21 w / v%, 0.22 w / v%, 0.23 w / v%, 0.24 w / v%, 0.25 w / v%, 0.26 w / v%, 0.27 w / v%, 0.28 w / v%, 0.29 w / v%, or 0.30 w / v% and any value in between.

[0060] Trypsin is a powerful enzyme with the ability to digest proteins. It can cleave chemical bonds in protein molecules and break down proteins into small fragments, making the proteins of cell membranes and organelles easy to degrade. However, trypsin is specific and only acts on specific types of amino acid linkages, which ensures that only proteins are digested while other cell components are not affected. The heart is a relatively dense organ wrapped by cardiomyocytes compared to other tissues and organs. Using an appropriate digestion time can maintain its integrity.

[0061] In one embodiment, after co-incubating the de-membraned embryo with a protease, a step of terminating digestion is further included;

[0062] Optionally, terminating digestion includes using fetal bovine serum at a concentration of 8 v / v% to 12 v / v%. For example, the concentration of fetal bovine serum is 8 v / v%, 9 v / v%, 10 v / v%, 11 v / v% or 12 v / v% and any value in between.

[0063] In one embodiment, de-membraning the embryo includes using one or both of physical treatment and chemical treatment.

[0064] In some embodiments, physical treatment includes directly using a puncturing tool.

[0065] In some embodiments, the puncturing tool includes a 200 μL pipette tip with a diameter of 0.4 mm to 0.5 mm. For example, the diameter of the pipette tip is 0.40 mm, 0.41 mm, 0.42 mm, 0.43 mm, 0.44 mm, 0.45 mm, 0.46 mm, 0.47 mm, 0.48 mm, 0.49 mm or 0.50 mm and any value in between.

[0066] In some embodiments, chemical treatment includes using a pronase de-membraning solution at a concentration of 0.8 mg / mL to 2 mg / mL. For example, the concentration of the pronase de-membraning solution is 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2.0 mg / mL and any value in between.

[0067] Pronase is a mixture of non-specific proteases extracted from Streptomyces griseus, which has broad proteolytic activity and can break down proteins into individual amino acids.

[0068] In some embodiments, a step of washing with a washing solution is further included before preparing the isolated heart;

[0069] In some embodiments, the washing solution includes Ringer's solution.

[0070] Ringer's Solution is an isotonic solution mainly used for in vitro culture of biological cells and biomedical research. Its components include sodium chloride, potassium chloride, calcium chloride, and sodium bicarbonate, which together simulate the ionic concentration and pH value of the internal environment in which cells are located.

[0071] On the other hand, this application provides the use of the zebrafish larval heart isolated by the above method for isolating the zebrafish larval heart in model animal research.

[0072] It can be understood that this research includes heart regeneration research. Zebrafish have a strong heart regeneration ability, and their hearts can recover function through the proliferation and regeneration of cardiomyocytes after injury. For example, through the apex resection model, zebrafish can completely recover the damaged heart tissue within 60 days. This model can be used to study the molecular mechanisms and cellular behaviors of heart regeneration, providing a theoretical basis for the treatment of human heart diseases.

[0073] The research also includes drug screening and toxicity assessment. The zebrafish model can be used for high-throughput screening of drugs with cardioprotective activity, while evaluating the cardiotoxicity of drugs. For example, by constructing a heart failure model, researchers can screen out compounds that can improve heart function.

[0074] The implementation schemes of this application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions in the following examples, the guidance given in this application should be preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in this field, or according to the conditions recommended by the manufacturers, or by referring to the experimental methods known in this field.

[0075] In the following specific examples, for the measurement parameters of raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0076] It should be understood that in various embodiments of this application, the magnitudes of the serial numbers of the above processes do not mean the sequence of execution. The execution sequence of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0077] Example 1

[0078] This example provides a method for isolating the zebrafish larval heart, including:

[0079] I. Preparation of transgenic line Tg(myl7:GFP) embryos:

[0080] Place the male and female Tg (myl7:GFP) transgenic fish in the aquarium one afternoon or evening in advance, separated by a partition. Remove the partition the next morning and let the zebrafish mate and lay eggs on their own. Collect the embryos and place them in an incubator for culture. Wait until a specific time to perform the heart isolation experiment.

[0081] 2. Isolation of zebrafish larvae heart:

[0082] 1. Select 24 hpf embryos.

[0083] 2. Select the embryos with positive cardiac fluorescence under a stereofluorescence microscope.

[0084] 3. Remove the embryonic membranes by chemically removing the membranes using Pronase demembraning solution at a working concentration of 1 mg / mL.

[0085] 4. Place 10 fry in a 1.5mL centrifuge tube, drain the embryo water, add 200μL of 0.25% trypsin, incubate for 15s, use a 200μL pipette, adjust the maximum range, and gently blow. After blowing, add 20μL of serum and gently blow to terminate trypsin digestion. Try to blow all embryos during the blowing process. Then transfer the suspension to a new culture dish with 1 / 3× Ringer's solution prepared in the culture dish to wash the suspension.

[0086] 3. Collection of isolated hearts:

[0087] When the suspension is placed under a stereofluorescence microscope, it can be seen that the heart tissue can be relatively intact and can be well separated from the embryo. Use a pipette to transfer the isolated heart to a new centrifuge tube. After collecting a certain number of isolated hearts, centrifuge at 12000g and 4℃ for 1 minute to remove the supernatant.

[0088] Next, you can conduct molecular biology experiments, extract RNA or protein for experiments, or perform high-throughput sequencing.

[0089] Example 2

[0090] This embodiment provides a method for isolating the heart of a zebrafish fry, comprising:

[0091] 1. Preparation of embryos of transgenic strain Tg (myl7:GFP):

[0092] Place the male and female Tg (myl7:GFP) transgenic fish in the aquarium one afternoon or evening in advance, separated by a partition. Remove the partition the next morning and let the zebrafish mate and lay eggs on their own. Collect the embryos and place them in an incubator for culture. Wait until a specific time to perform the heart isolation experiment.

[0093] 2. Isolation of zebrafish larvae heart:

[0094] 1. Select embryos at 36 hpf.

[0095] 2. Select the embryos with positive cardiac fluorescence under a stereofluorescence microscope.

[0096] 3. Remove the embryonic membranes by chemically removing the membranes using Pronase demembraning solution at a working concentration of 1 mg / mL.

[0097] 4. Place 10 fry in a 1.5mL centrifuge tube, drain the embryo water, add 200μL of 0.25% trypsin, incubate for 30s, use a 200μL pipette, adjust the maximum range, and gently blow. After blowing, add 20μL of serum and gently blow to terminate trypsin digestion. Try to blow all embryos during the blowing process. Then transfer the suspension to a new culture dish with 1 / 3× Ringer's solution prepared in the culture dish to wash the suspension.

[0098] 3. Collection of isolated hearts:

[0099] When the suspension is placed under a stereofluorescence microscope, it can be seen that the heart tissue can be relatively intact and can be well separated from the embryo. Use a pipette to transfer the isolated heart to a new centrifuge tube. After collecting a certain number of isolated hearts, centrifuge at 12000g and 4℃ for 1 minute to remove the supernatant.

[0100] Next, you can conduct molecular biology experiments, extract RNA or protein for experiments, or perform high-throughput sequencing.

[0101] Example 3

[0102] 1. Preparation of embryos of transgenic strain Tg (myl7:GFP):

[0103] Place the male and female Tg (myl7:GFP) transgenic fish in the aquarium one afternoon or evening in advance, separated by a partition. Remove the partition the next morning and let the zebrafish mate and lay eggs on their own. Collect the embryos and place them in an incubator for culture. Wait until a specific time to perform the heart isolation experiment.

[0104] 2. Isolation of zebrafish larvae heart:

[0105] 1. Select embryos at 48 hpf.

[0106] 2. Select the embryos with positive cardiac fluorescence under a stereofluorescence microscope.

[0107] 3. Remove the embryonic membranes by chemically removing the membranes using Pronase demembraning solution at a working concentration of 1 mg / mL.

[0108] 4. Place 10 fry in a 1.5mL centrifuge tube, drain the embryo water, add 200μL of 0.25% trypsin, incubate for 60s, use a 200μL pipette, adjust the maximum range, and gently blow. After blowing, add 20μL of serum and gently blow to terminate trypsin digestion. Try to blow all embryos during the blowing process. Then transfer the suspension to a new culture dish with 1 / 3× Ringer's solution prepared in the culture dish to wash the suspension.

[0109] 3. Collection of isolated hearts:

[0110] When the suspension is placed under a stereofluorescence microscope, it can be seen that the heart tissue can be relatively intact and can be well separated from the embryo. Use a pipette to transfer the isolated heart to a new centrifuge tube. After collecting a certain number of isolated hearts, centrifuge at 12000g and 4℃ for 1 minute to remove the supernatant.

[0111] Next, you can conduct molecular biology experiments, extract RNA or protein for experiments, or perform high-throughput sequencing.

[0112] Example 4

[0113] 1. Preparation of embryos of transgenic strain Tg (myl7:GFP):

[0114] Place the male and female Tg (myl7:GFP) transgenic fish in the aquarium one afternoon or evening in advance, separated by a partition. Remove the partition the next morning and let the zebrafish mate and lay eggs on their own. Collect the embryos and place them in an incubator for culture. Wait until a specific time to perform the heart isolation experiment.

[0115] 2. Isolation of zebrafish larvae heart:

[0116] 1. Select embryos at 72 hpf.

[0117] 2. Select the embryos with positive cardiac fluorescence under a stereofluorescence microscope.

[0118] 3. Remove the embryonic membranes by chemically removing the membranes using Pronase demembraning solution at a working concentration of 1 mg / mL.

[0119] 4. Place 10 fry in a 1.5mL centrifuge tube, drain the embryo water, add 200μL of 0.25% trypsin, incubate for 60s, use a 200μL pipette, adjust the maximum range, and gently blow. After blowing, add 20μL of serum and gently blow to terminate trypsin digestion. Try to blow all embryos during the blowing process. Then transfer the suspension to a new culture dish with 1 / 3× Ringer's solution prepared in the culture dish to wash the suspension.

[0120] III. Collection of ex vivo hearts:

[0121] When the suspension is placed under a stereoscopic fluorescence microscope, it can be seen that the heart tissue can be relatively completely retained and can also be well separated from the embryo body. Use a pipette to aspirate the ex vivo heart into a new centrifuge tube. After collecting a certain number of ex vivo hearts, centrifuge at 12,000 g and 4 °C for 1 min to remove the supernatant.

[0122] Next, molecular biology experiments can be carried out, such as extracting RNA or protein for experiments, or performing high-throughput sequencing.

[0123] Verification and analysis of digestion time:

[0124] Since trypsin is a strong enzyme for digesting proteins, in order to prevent over-digestion, this application further compares the different digestion times required for the hearts at different developmental stages to be completely dissociated without being over-digested into cell clusters.

[0125] The digestion times compared in this application are different time points such as 15 s, 30 s, 1 min, 1.5 min, 2 min, etc. Take pictures to observe the separation degree and integrity of the heart.

[0126] 1. At the 24 hpf stage, the embryo has just developed into the shape of a juvenile fish, and the heart is located below the head and on the left side of the embryo body. For embryos at this stage, this application selects trypsin digestion times of 15 s and 30 s.

[0127] As Figure 1 shown, the results show that: after trypsin digestion for 15 s, it is found that the heart tissue can be relatively completely retained and can also be well separated from the embryo body. After trypsin digestion for 30 s, the heart tissue is over-digested, the heart tissue is severely dispersed, and the degree of fragmentation is relatively high.

[0128] Therefore, at the 24 hpf stage, this application selects a trypsin digestion time of 15 s.

[0129] 2. At the 36 hpf stage, the heart has started to loop, but the heart is still located below the left head. For this stage, this application selects trypsin digestion times of 15 s, 30 s, and 1 min.

[0130] As Figure 2 shown, the results show that: after trypsin digestion for 15 s, the heart cannot be well separated from the embryo body. After digestion for 30 s, it is found that the heart tissue can be relatively completely retained and can also be well separated from the embryo body. However, after trypsin digestion for 1 min, fragments appear in the heart tissue.

[0131] Therefore, at the 36 hpf stage, this application selects a trypsin digestion time of 30 s.

[0132] 3. At the 48 hpf stage, the heart has already been located in front of the yolk. For this stage, three time points of 30 s, 1 min, and 2 min were selected in this application.

[0133] As Figure 3 shown, it was found that: after trypsin digestion for 30 s, the heart could not be well separated from the embryo body, and the digestion time was insufficient. When trypsin was digested for 1 min, the heart tissue could be well separated, and there were also fewer dispersed heart cells. The heart was relatively complete, and the tissue was also relatively complete. When trypsin was digested for 2 min, the digestion time was too long, resulting in the separation of heart cells and the destruction of the integrity of the heart.

[0134] Therefore, at the 48 hpf stage, this application selects a trypsin digestion time of 1 min.

[0135] 4. At the 72 hpf stage, heart morphogenesis has ended. For this stage, three time points of 30 s, 1 min, and 2 min were selected in this application.

[0136] As Figure 4 shown, it was found that: after trypsin digestion for 30 s, the heart could not be well separated from the embryo body, and the digestion time was insufficient. When trypsin was digested for 1 min, the heart tissue could be well separated, and there were also fewer dispersed heart cells. The heart was relatively complete, and the tissue was also relatively complete. When trypsin was digested for 2 min, the digestion time was too long, resulting in the separation of heart cells and the destruction of the integrity of the heart.

[0137] Therefore, at the 72 hpf stage, this application selects a trypsin digestion time of 1 min.

[0138] The above-described embodiments only represent several implementation manners of this application, which are convenient for understanding the technical solutions of this application specifically and in detail, but should not be construed as limiting the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several deformations and improvements can be made, and these all belong to the protection scope of this application. In addition, it should be understood that after reading the above teachings of this application, those skilled in the art can make various changes or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided in this application are all within the protection scope of the appended claims of this application. Therefore, the protection scope of this application patent should be based on the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for isolating the heart of a zebrafish fry, characterized in that: include: Providing zebrafish embryos, and performing a demembranating process on the embryos; The embryos after demembranation were digested with trypsin; as well as, The digestion product was blown to prepare the isolated zebrafish larvae heart; The development period of the zebrafish embryo is 24 hpf to 72 hpf, and the co-incubation time is 15 s to 62 s.

2. The method for isolating the heart of a zebrafish fry according to claim 1, characterized in that: The development period of the zebrafish embryos is 24 hpf to 26 hpf, and the co-incubation time is 14 s to 16 s; When the developmental period of the zebrafish embryo is 35 hpf to 37 hpf, the co-incubation time is 28 s to 32 s; When the developmental period of the zebrafish embryo is 46 hpf to 50 hpf, the co-incubation time is 58 s to 62 s; When the developmental period of the zebrafish embryo is 70 hpf to 74 hpf, the co-incubation time is 58 s to 62 s.

3. The method for isolating the heart of a zebrafish fry according to claim 1, characterized in that: The components of the trypsin include one or more of lipase, nuclease, polysaccharide and protease; Optionally, the trypsin is trypsin-EDTA.

4. The method for isolating the zebrafish fry heart according to claim 3, characterized in that: The concentration of the trypsin is 0.20 w / v% to 0.30 w / v%.

5. The method for isolating the heart of a juvenile zebrafish according to any one of claims 1 to 4, characterized in that: The step of digesting the embryo after the demembranation treatment with trypsin also includes the step of terminating the digestion; Optionally, terminating the digestion comprises using fetal bovine serum at a concentration of 8 v / v% to 12 v / v%.

6. The method for isolating the heart of a zebrafish fry according to any one of claims 1 to 4, characterized in that: The demolding treatment includes one or both of physical treatment and chemical treatment.

7. The method for isolating the heart of a zebrafish fry according to claim 6, characterized in that: The physical treatment includes using a puncture tool; optionally, the puncture tool includes a needle with a diameter of 0.4 mm to 0.5 mm.

8. The method for isolating the heart of a zebrafish fry according to claim 6, characterized in that: Chemical treatment includes the use of pronase stripping solution.

9. The method for isolating the zebrafish fry heart according to claim 8, characterized in that: The concentration of the pronase stripping solution is 0.8 mg / mL to 1.2 mg / mL.

10. The method for isolating the heart of a young zebrafish according to any one of claims 1 to 4 and 7 to 9, characterized in that: The method further comprises the step of washing with a washing solution before preparing the separated zebrafish larvae heart; Optionally, the cleaning solution comprises Ringer's solution.

Images