CN103091144A - Method for preparing chromosomes through large-scale barbel fish nephrocyte in-vivo culture - Google Patents
Method for preparing chromosomes through large-scale barbel fish nephrocyte in-vivo culture Download PDFInfo
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- CN103091144A CN103091144A CN2013100339181A CN201310033918A CN103091144A CN 103091144 A CN103091144 A CN 103091144A CN 2013100339181 A CN2013100339181 A CN 2013100339181A CN 201310033918 A CN201310033918 A CN 201310033918A CN 103091144 A CN103091144 A CN 103091144A
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Abstract
The invention discloses a method for preparing chromosomes through large-scale barbel fish nephrocyte in-vivo culture and relates to a method for preparing fish chromosomes. The invention aims to solve the technical problem of the method for preparing the chromosomes of the large-scale barbel fish. The method comprises the following steps of: performing living body injection, namely injecting phytohaemagglutinin (PHA) into an abdominal cavity of the living body twice, and injecting colchicine; 2, sampling, namely washing head-kidney in NaCl normal saline, and collecting cells; 3, performing hypotension by using a mixed solution of sodium citrate and KCl; 4, performing cryofixation by using a Carnoy's fixing solution; 5, dropping through a cold drop method; and 6, staining through Giemsa staining solution, and obtaining the chromosomes of the large-scale barbel fish. The method is easy and convenient to operate and stable in result, and the large-scale barbel fish metacinesis specimen which is complete in number, good in dispersion and clear in shape can be obtained. The method is applied to the field of preparation of fish chromosomes.
Description
Technical field
The present invention relates to the preparation method of Fishes Chromosomes.
Background technology
Large squama Barb (Barbuscapito) genus Cyprinidae (Cyprinidae), Barbinae (Barbinae), Barb belong to (Barbodes), originate in the Saltwater Sea of Uzbekistan.It has the merits such as feeding habits are wide, fast growth, delicious meat, Salt And Alkali Tolerance, strong adaptability, its maximum individual long 70cm, heavy 12kg, it is local important large-scale economic fish (Nico Li Siji work, Miu Xuezu, Lin Fushen, Tian Mingcheng translates. minute door ichthyology [M]. and Beijing: Higher Education Publishing House, 1958:186-189.).Heilungkiang aquatic products research institute introduced China with this fish in 2003, was intended to increase inland saline-alkali water fish culture kind, to promote the development and utilization of saline-alkali water.
Chromosome is the main carriers of inhereditary material, chromosome number and karyotype are one of main contents of biological cytogenetics research, chromosome evolutionary process, categorizing system and evolutionary relationship to understanding and exploration biology are significant, also can be the fish genetic breeding cytogenetics foundation is provided.The chromosome preparation is a basic technology in cytogenetical study, and obtaining fast, easily the chromosome result that form is good, di is high is prerequisite and the basis of carrying out karyotyping.At present, the Fishes Chromosomes preparation method mainly is divided into cultivation and cell culture method three major types in direct method, body, although preparation principle is basic identical, due to the difference of different Fish Tissue structures and the difference of disposal route, the quality and quantity of the metaphase in cell division phase that obtains also is not quite similar.Therefore, the chromosome preparation condition of every kind of fish maturation still needs constantly to grope could set up with repetition test.At present, relate to the interior preparation method of cultivation of the chromosomal body of large squama Barb and yet there are no report.
Summary of the invention
The invention provides and cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body.
Cultivating the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body of the present invention carries out according to the following steps:
One, live body injection: weigh after with 5～10ppm Phenoxyethanol, large squama Barb fish being anaesthetized, live body lumbar injection phytohemagglutinin (PHA) for the first time, dosage is 7～12 μ g: 1g with the ratio of large squama Barb fish body weight, live body lumbar injection phytohemagglutinin (PHA) for the second time after 16～20h, dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, after effect 4～6h, plastic injection quality concentration is 0.1%～0.2% colchicine, dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, and the reaction time is 2～4h;
Two, sampling: the large squama Barb fish that step 1 is processed is cut the gill, bloodletting 10～15min in water, then taking out head-kidney washs in mass concentration is 0.7%～0.9% NaCl physiological saline, with operating scissors shred become muddiness to physiological saline till, collect the upper strata cell suspension in centrifuge tube with suction pipe after standing 3～5min, be centrifugal 5～10min under the condition of 1000～1500r/min at rotating speed, abandon supernatant, collecting cell;
Three, hypotonic: as to add hypotonic medium in the cell of step 2 collection, blow and beat to the cell Uniform Dispersion room temperature reaction 40～60min with suction pipe; Wherein hypotonic medium is that 0.1%～0.5% KCl solution and mass concentration are that 0.1%～0.5% sodium citrate solution is 1: 1～2 to mix by volume by mass concentration;
Four, fixing: the cell that step 3 is hypotonic is centrifugal 5～10min under the condition of 1000～2000r/min at rotating speed, after staying the supernatant of 0.3～0.5mL to blow and beat into cell suspension, adds the Ka Nuoshi immobile liquid to fix 20～30min;
Five, after the operation 1～2 time of repeating step four, centrifugal 5～10min, abandon supernatant under the rotating speed of 1000～1500r/min, and sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid and is placed on-18～-20 ℃ of lower freeze overnight;
Six, cold sheet: abandon supernatant after centrifugal the freezing cell suspension of step 5, add the Ka Nuoshi immobile liquid of 2～3mL to mix, get the clean slide that is immersed in precooling in the frozen water mixed liquor, disperse to drip upper 2～3 cell suspensions, immediately microslide is crossed 3～5 times above spirit lamp flame, make Chromosome spread and expansion, then dry under room temperature;
Seven, dyeing: be 10%～15% Jim Sa (Giemsa) dyeing liquor, the 30～50min that dyes with the Chromosome glass slide volume fraction of step 6 drying, tap water rinses, complete the chromosome specimen preparation after natural drying, namely obtain large squama Barb fish chromosome.
The present invention includes following beneficial effect:
1, the present invention adopts double injection phytohemagglutinin (PHA), and dosage is 7～12 μ g: 1g with the ratio of large squama Barb fish body weight for the first time, and dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight for the second time, makes fish somatic cell proliferation effect better;
2, the injected dose of colchicine of the present invention is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, and the reaction time is 2～4h, can preferably cell division be rested on mid-term, and the acquisition form is better, the chromosome metacinesis phase of clear picture;
3, the present invention's employing mass concentration when the processing head-kidney is organized is 0.7% NaCl physiological saline, near the osmotic pressure of nephrocyte, avoids cell to be subjected to outer liquid osmotic pressure influence broken;
4, hypotonic medium composition of the present invention is that mass concentration is that 0.2% KCl and mass concentration are 0.2% sodium citrate mixed solution, the advantage of KCl is that the chromosome profile is clear, but dyeability is strong, the advantage of sodium citrate is to have good pH to regulate and shock-absorbing capacity, both mix use, can keep the stable of chromosome surrounding environment in the hypotonic process of cell, be beneficial to chromosome morphology and keep complete;
5, the present invention is easy and simple to handle, and result is stable, can obtain that number is complete, good dispersion, form large squama Barb fish chromosome metacinesis phase sample clearly.
Description of drawings
Fig. 1 is the large squama Barb fish chromosome metacinesis phase of test one preparation.
Embodiment
Embodiment one: cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body of present embodiment and carry out according to the following steps:
One, live body injection: weigh after with 5～10ppm Phenoxyethanol, large squama Barb fish being anaesthetized, live body lumbar injection phytohemagglutinin (PHA) for the first time, dosage is 7～12 μ g: 1g with the ratio of large squama Barb fish body weight, live body lumbar injection phytohemagglutinin (PHA) for the second time after 16～20h, dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, after effect 4～6h, plastic injection quality concentration is 0.1%～0.2% colchicine, dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, and the reaction time is 2～4h;
Two, sampling: the large squama Barb fish that step 1 is processed is cut the gill, bloodletting 10～15min in water, then taking out head-kidney washs in mass concentration is 0.7%～0.9% NaCl physiological saline, with operating scissors shred become muddiness to physiological saline till, collect the upper strata cell suspension in centrifuge tube with suction pipe after standing 3～5min, be centrifugal 5～10min under the condition of 1000～1500r/min at rotating speed, abandon supernatant, collecting cell;
Three, hypotonic: as to add hypotonic medium in the cell of step 2 collection, blow and beat to the cell Uniform Dispersion room temperature reaction 40～60min with suction pipe; Wherein hypotonic medium is that 0.1%～0.5% KCl solution and mass concentration are that 0.1%～0.5% sodium citrate solution is 1: 1～2 to mix by volume by mass concentration;
Four, fixing: the cell that step 3 is hypotonic is centrifugal 5～10min under the condition of 1000～2000r/min at rotating speed, after staying the supernatant of 0.3～0.5mL to blow and beat into cell suspension, adds the Ka Nuoshi immobile liquid to fix 20～30min;
Five, after the operation 1～2 time of repeating step four, centrifugal 5～10min, abandon supernatant under the rotating speed of 1000～1500r/min, and sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid and is placed on-18～-20 ℃ of lower freeze overnight;
Six, cold sheet: abandon supernatant after centrifugal the freezing cell suspension of step 5, add the Ka Nuoshi immobile liquid of 2～3mL to mix, get the clean slide that is immersed in precooling in the frozen water mixed liquor, disperse to drip upper 2～3 cell suspensions, immediately microslide is crossed 3～5 times above spirit lamp flame, make Chromosome spread and expansion, then dry under room temperature;
Seven, dyeing: be 10%～15% Jim Sa (Giemsa) dyeing liquor, the 30～50min that dyes with the Chromosome glass slide volume fraction of step 6 drying, tap water rinses, complete the chromosome specimen preparation after natural drying, namely obtain large squama Barb fish chromosome.
Present embodiment comprises following beneficial effect:
1, present embodiment adopts double injection phytohemagglutinin (PHA), dosage is 7～12 μ g: 1g with the ratio of large squama Barb fish body weight for the first time, dosage is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight for the second time, makes fish somatic cell proliferation effect better;
2, the injected dose of the colchicine of present embodiment is 5～10 μ g: 1g with the ratio of large squama Barb fish body weight, and the reaction time is 2～4h, can preferably cell division be rested on mid-term, and the acquisition form is better, the chromosome metacinesis phase of clear picture;
3, present embodiment employing mass concentration when the processing head-kidney is organized is 0.7% NaCl physiological saline, near the osmotic pressure of nephrocyte, avoids cell to be subjected to outer liquid osmotic pressure influence broken;
4, the hypotonic medium composition of present embodiment is that mass concentration is that 0.2% KCl and mass concentration are 0.2% sodium citrate, the advantage of KCl is that the chromosome profile is clear, but dyeability is strong, the advantage of sodium citrate is to have good pH to regulate and shock-absorbing capacity, both mix use, can keep the stable of chromosome surrounding environment in the hypotonic process of cell, be beneficial to chromosome morphology and keep complete;
5, present embodiment is easy and simple to handle, and result is stable, can obtain that number is complete, good dispersion, form large squama Barb fish chromosome metacinesis phase sample clearly.
Embodiment two: what present embodiment was different from collective embodiment one is: be centrifugal 5min under the condition of 1000r/min at rotating speed in step 2.Other is identical with embodiment one.
Embodiment three: what present embodiment was different from collective embodiment one or two is: be centrifugal 5min under the condition of 1000r/min at rotating speed in step 4.Other is identical with embodiment one or two.
Embodiment four: what present embodiment was different from one of collective embodiment one to three is: add the Ka Nuoshi immobile liquid to blow and beat into after cell suspension fixedly 20min in step 4.Other is identical with one of embodiment one to three.
Embodiment five: what present embodiment was different from one of collective embodiment one to four is: in step 5 under the rotating speed of 1000r/min centrifugal 5min.Other is identical with one of embodiment one to four.
Embodiment six: what present embodiment was different from one of collective embodiment one to five is: in step 5, sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid and is placed on-18 ℃ of lower freeze overnight.Other is identical with one of embodiment one to five.
Embodiment seven: what present embodiment was different from one of collective embodiment one to six is: be 10% Jim Sa (Giemsa) dyeing liquor dyeing 30min with volume fraction in step 7.Other is identical with one of embodiment one to six.
By following verification experimental verification beneficial effect of the present invention:
Test one: cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body of this test and realize according to the following steps:
One, live body injection: weigh after with the 5ppm Phenoxyethanol, large squama Barb fish being anaesthetized, live body lumbar injection phytohemagglutinin (PHA) for the first time, dosage is 7 μ g: 1g with the ratio of large squama Barb fish body weight, live body lumbar injection phytohemagglutinin (PHA) for the second time after 16h, dosage is 5 μ g: 1g with the ratio of large squama Barb fish body weight, after effect 4h, plastic injection quality concentration is 0.1% colchicine, and dosage is 5 μ g: 1g with the ratio of large squama Barb fish body weight, and the reaction time is 2h;
Two, sampling: the large squama Barb fish that step 1 is processed is cut the gill, bloodletting 10min in water, then taking out head-kidney washs in mass concentration is 0.7% NaCl physiological saline, with operating scissors shred become muddiness to physiological saline till, collect the upper strata cell suspension in centrifuge tube with suction pipe after standing 3min, be centrifugal 5min under the condition of 1000r/min at rotating speed, abandon supernatant, collecting cell;
Three, hypotonic: as to add hypotonic medium in the cell of step 2 collection, blow and beat to the cell Uniform Dispersion room temperature reaction 45min with suction pipe; Wherein hypotonic medium is that 0.2% KCl solution and mass concentration are that 0.2% sodium citrate solution is to mix at 1: 1 by volume by mass concentration;
Four, fixing: the cell that step 3 is hypotonic is centrifugal 5min under the condition of 1000r/min at rotating speed, after staying the supernatant of 0.5mL to blow and beat into cell suspension, adds fixedly 20min of Ka Nuoshi immobile liquid;
Five, after the operation 1 time of repeating step four, centrifugal 5min, abandon supernatant under the rotating speed of 1000r/min, and sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid and is placed on-18 ℃ of lower freeze overnight;
Six, cold sheet: abandon supernatant after centrifugal the freezing cell suspension of step 5, add the Ka Nuoshi immobile liquid of 2mL to mix, get the clean slide that is immersed in precooling in the frozen water mixed liquor, disperse to drip upper 3 cell suspensions, immediately microslide is crossed 3 times above spirit lamp flame, make Chromosome spread and expansion, then dry under room temperature;
Seven, dyeing: be 10% Jim Sa (Giemsa) dyeing liquor dyeing 30min with the Chromosome glass slide volume fraction of step 6 drying, tap water rinses, and completes chromosome specimen after natural drying to prepare, and namely obtains large squama Barb fish chromosome.
Double injection phytohemagglutinin (PHA) is adopted in this test, and dosage is 7 μ g: 1g with the ratio of large squama Barb fish body weight for the first time, and dosage is 5 μ g: 1g with the ratio of large squama Barb fish body weight for the second time, makes fish somatic cell proliferation effect better;
When this test is organized at the processing head-kidney, the employing mass concentration is 0.7% NaCl physiological saline, near the osmotic pressure of nephrocyte, avoids cell to be subjected to outer liquid osmotic pressure influence broken;
The hypotonic medium composition of this test is that mass concentration is that 0.2% KCl and mass concentration are 0.2% sodium citrate, the advantage of KCl is that the chromosome profile is clear, but dyeability is strong, the advantage of sodium citrate is to have good pH to regulate and shock-absorbing capacity, both mix use, can keep the stable of chromosome surrounding environment in the hypotonic process of cell, be beneficial to chromosome morphology and keep complete;
The chromosome slide of dyeing is placed in the microscopically observation, the large squama Barb fish chromosome metacinesis of this test preparation mutually as shown in Figure 1, as can be seen from Figure 1 this test obtained that number is complete, good dispersion, form large squama Barb chromosome metacinesis phase sample clearly.
Claims (7)
1. cultivate the chromosomal method of preparation in a large squama Barb fish nephrocyte body, it is characterized in that cultivating in large squama Barb fish nephrocyte body the chromosomal method of preparation and carry out according to the following steps:
One, live body injection: weigh after with 5～10ppm Phenoxyethanol, large squama Barb fish being anaesthetized, live body lumbar injection phytohemagglutinin (PHA) for the first time, dosage is 7～12 μ g: 1g with the ratio of body weight, live body lumbar injection phytohemagglutinin (PHA) for the second time after 16～20h, dosage is 5～10 μ g: 1g with the ratio of body weight, after effect 4～6h, plastic injection quality concentration is 0.1%～0.2% colchicine, dosage is 5～10 μ g: 1g with the ratio of body weight, and the reaction time is 2～4h;
Two, sampling: the large squama Barb fish that step 1 is processed is cut the gill, bloodletting 10～15min in water, then taking out head-kidney washs in mass concentration is 0.7%～0.9% NaCl physiological saline, with operating scissors shred become muddiness to physiological saline till, collect the upper strata cell suspension in centrifuge tube with suction pipe after standing 3～5min, be centrifugal 5～10min under the condition of 1000～1500r/min at rotating speed, abandon supernatant, collecting cell;
Three, hypotonic: as to add hypotonic medium in the cell of step 2 collection, blow and beat to the cell Uniform Dispersion room temperature reaction 40～60min with suction pipe; Wherein hypotonic medium is that 0.1%～0.5% KCl solution and mass concentration are that 0.1%～0.5% sodium citrate solution is 1: 1～2 to mix by volume by mass concentration;
Four, fixing: the cell that step 3 is hypotonic is centrifugal 5～10min under the condition of 1000～2000r/min at rotating speed, after staying the supernatant of 0.3～0.5mL to blow and beat into cell suspension, adds the Ka Nuoshi immobile liquid to fix 20～30min;
Five, after the operation 1～2 time of repeating step four, centrifugal 5～10min, abandon supernatant under the rotating speed of 1000～1500r/min, and sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid and is placed on-18～-20 ℃ of lower freeze overnight;
Six, cold sheet: abandon supernatant after centrifugal the freezing cell suspension of step 5, add the Ka Nuoshi immobile liquid of 2～3mL to mix, get the clean slide that is immersed in precooling in the frozen water mixed liquor, disperse to drip upper 2～3 cell suspensions, immediately microslide is crossed 3～5 times above spirit lamp flame, make Chromosome spread and expansion, then dry under room temperature;
Seven, dyeing: be Giemsa staining liquid dyeing 30～50min of 10%～15% with the Chromosome glass slide volume fraction of step 6 drying, tap water rinses, and completes chromosome specimen after natural drying to prepare, and namely obtains large squama Barb fish chromosome.
2. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that in step 2 that at rotating speed be centrifugal 5min under the condition of 1000r/min.
3. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that in step 4 that at rotating speed be centrifugal 5min under the condition of 1000r/min.
4. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that adding in step 4 the Ka Nuoshi immobile liquid to blow and beat into after cell suspension fixedly 20min.
5. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that in step 5 centrifugal 5min under the rotating speed of 1000r/min.
6. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that in step 5, sediment is blown and beaten into cell suspension with the Ka Nuoshi immobile liquid to be placed on-18 ℃ of lower freeze overnight.
7. cultivate the chromosomal method of preparation in a kind of large squama Barb fish nephrocyte body according to claim 1, it is characterized in that in step 7 that with volume fraction be 10% the Giemsa staining liquid 30min that dyes.
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Cited By (6)
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| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
| CN109030131A (en) * | 2018-06-12 | 2018-12-18 | 中国水产科学研究院黑龙江水产研究所 | A kind of refined dragonfish method of chromosome preparation |
| CN111323286A (en) * | 2020-03-16 | 2020-06-23 | 湖南师范大学 | Method for preparing chromosome specimen by using small molecule inhibitor SP600125 |
| CN111793671A (en) * | 2020-07-22 | 2020-10-20 | 中国农业大学 | Optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes |
| CN112577803A (en) * | 2020-12-10 | 2021-03-30 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
| CN115046808A (en) * | 2022-07-20 | 2022-09-13 | 浙江省淡水水产研究所 | Minimally invasive blood extraction method and chromosome flaking method for turtle animals |
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| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
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| CN111793671A (en) * | 2020-07-22 | 2020-10-20 | 中国农业大学 | Optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes |
| CN112577803A (en) * | 2020-12-10 | 2021-03-30 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
| CN112577803B (en) * | 2020-12-10 | 2021-11-09 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
| CN115046808A (en) * | 2022-07-20 | 2022-09-13 | 浙江省淡水水产研究所 | Minimally invasive blood extraction method and chromosome flaking method for turtle animals |
| CN115046808B (en) * | 2022-07-20 | 2023-09-01 | 浙江省淡水水产研究所 | Minimally invasive blood sampling method and chromosome slicing method for soft-shelled turtle animals |
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Application publication date: 20130508 |