CN102509504A - Method for preparing chromosome specimen with sepia esculenta embryonic cell - Google Patents
Method for preparing chromosome specimen with sepia esculenta embryonic cell Download PDFInfo
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- CN102509504A CN102509504A CN2011102841169A CN201110284116A CN102509504A CN 102509504 A CN102509504 A CN 102509504A CN 2011102841169 A CN2011102841169 A CN 2011102841169A CN 201110284116 A CN201110284116 A CN 201110284116A CN 102509504 A CN102509504 A CN 102509504A
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Abstract
A method for preparing a chromosome specimen with a sepia esculenta embryonic cell, which includes the operating procedures of stripping, pre-treating, carrying out hypotonic treatment, fixing, dissociating, dripping, dyeing and washing, and is characterized in that the dripping adopts thermal dripping at the temperature ranging from 40 DEG C to 60 DEG C, the height in the dripping ranges from 1.8mto 2m, and absorbent paper is used for absorbing redundant liquid at a middle position of each liquid drop after each liquid drop is dripped. By means of the thermal dripping, the temperature of the dripping and the height in dripping are reasonably controlled, the chromosome production time is shortened, the chromosome production effect is improved, and the problems of difficulty in mastering of cell rupture degree, chromosome loss, poor chromosome morphology and the like when the chromosome specimen is prepared are solved. The method is used for the chromosome production with the sepia esculenta embryonic cell, so that the good chromosome specimen is obtained, and the chromosome is multiple in phases in metakinesis, clear in morphology and fine in dispersion. Specific effects are indicated in a figure 1.
Description
Technical field:
The invention belongs to the cell biology field, concrete relate to a kind of method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen.
Background technology:
The preparation manipulation of chromosome specimen is simple, cheap, the low characteristics of experiment site requirements; It is traditional biogenetics investigative technique always; Chromosome specimen preparation comprises: pre-treatment, hypotonic processing, fix, dissociate, drip sheet, step such as dye and develop a film, obtained widespread use in many fields of biogenetics.But owing to the ripe individuality of golden cuttlefish obtain difficulty, cost higher, make reason such as chromosome specimen weak effect, utilize ripe individual preparation chromosome to seem unsatisfactory.The embryo is as one of material for preparing chromosome specimen; Obtain relatively easy, cost is low; Be widely used in the preparation of chromosome specimen; Prepare in the chromosome specimen process the normal cold conventional sheet method that adopt the golden cuttlefish embryo, the dry required time of slide is long, be prone to repeat a sheet, the cell difficulty is broken, and influences chromosome observation.
Summary of the invention:
The technical matters that the present invention will solve provides and a kind ofly can be good at utilizing the golden cuttlefish embryo to prepare the method for chromosome specimen; This method is dripped the sheet method through heat; Height when control is rationally dripped the temperature of sheet and dripped sheet; Shortened the film-making time, improved the chromosome sectioning effect, the cell rupture degree is grasped aspect problems such as difficulty, chromosome diminution, chromosome morphology difference when having solved chromosome specimen and preparing.
The present invention realizes by following technical scheme:
A kind of method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen, its running program are stripping, pre-treatment, hypotonic processing, fix, dissociate, drip sheet, dye and develop a film; Described sheet: adopt heat to drip the sheet method, 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, whenever drip off one after with thieving paper in the drop centre position absorption excess liquid.
As a kind of optimized technical scheme of the present invention, the method that above-mentioned golden cuttlefish embryonic cell prepares chromosome specimen may further comprise the steps:
Stripping: use fixedly ovum of tweezers, needle egg membrane with dissecting needle; Both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo;
Pre-treatment: the embryo behind the stripping adopts colchicine to carry out pre-treatment, handles 30min in the colchicine solution with embryonic cell immersion 0.04%;
Hypotonic processing: adopt concentration be 5% KCl as hypotonic medium, handle 45min, remove clean colchicine before the hypotonic processing;
Fixing: adopt Ka Nuoshi liquid (methyl alcohol: glacial acetic acid=3: 1, volume ratio) fixing, 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times, need remove half hypotonic medium before adding immobile liquid for the first time;
Dissociate: adopting concentration is the 50% glacial acetic acid 45min that dissociates, and removes clean immobile liquid before dissociating;
Drip sheet: adopt heat to drip the sheet method, 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, whenever drip off one after with thieving paper in the drop centre position absorption excess liquid;
Dyeing: adopt the dyeing of 10%Giemsa dye liquor, i.e. Giemsa stoste 1ml, the phosphate buffer 9ml of PH 6.8, dyeing time 30min;
Develop a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping, and the slide lower end becomes 30 ° with surface level, and the slide frosting contacts with current, and current just become wire to drip, slowly flushing, and the water to flushing no longer has till the color.
The beneficial effect of the present invention and prior art contrast is:
The present invention adopts heat to drip the sheet method, drips 40 ℃-60 ℃ of sheet temperature, and in this temperature range, cell can finely break, and guarantees that simultaneously drop can be too high and dry not too fast because of temperature, helps drop and extends; Drop is through the height of drop of 1.8m-2m, and cell rupture when contacting with slide can fully discharge chromosome; After drop drips on the slide, absorb excess liquid in the drop centre position with thieving paper immediately, make the concentrated mutually drop edge that is distributed in of division help observing; And droplet drying is very fast after absorbing unnecessary liquid; Clear-cut helps holding the direction of droplets fall, avoids repeating dripping a sheet.
Description of drawings:
Fig. 1, golden cuttlefish chromosome metacinesis phase
Embodiment:
Combine accompanying drawing to be described in detail technical method of the present invention through concrete instance below:
The golden cuttlefish embryonic cell is made the chromosome specimen step and is comprised: stripping, pre-treatment, hypotonic processing, fix, dissociate, drip sheet and dyeing.The present invention prepares in the process at chromosome specimen, has carried out the confirming and the improvement of the step of developing a film of the improvement of confirming, drip the sheet method, dyeing time of the time of confirming, dissociate of the confirming of selection and hypotonic time, the fixed routine of the confirming of the confirming of stripping method, pre-treatment time, hypotonic medium.
Confirming of stripping method: embryonic cell prepares chromosome specimen and generally is used for fish, because the ovum of fish is generally all less, can't carries out stripping and handle and obtain the embryo, so all adopt the mode of direct pre-treatment to carry out.But the golden cuttlefish egg membrane is thick, and the colchicine difficulty is soaked into, and ovum helps stripping acquisition embryo greatly.Use fixedly ovum of tweezers during stripping, needle egg membrane with dissecting needle, both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo, have shortened the pre-treatment time that time goes on foot greatly.
The pre-treatment time: the samples using colchicine carries out pre-treatment, and the pre-treatment time, the too short or long chromosome morphology that all can make was unfavorable for observing, and handled 30min in the colchicine solution of present embodiment with embryonic cell immersion 0.04%.
Confirming of the selection of hypotonic medium and hypotonic time: conventional method adopts filtering sea as hypotonic medium, because concentration of seawater confirms to cause the hypotonic time uncertain than difficulty, we adopt KCl as hypotonic medium, and concentration is 5%, handles 45min.Remove clean colchicine before the hypotonic processing.
Confirming of fixed routine: after finding that in experimentation immobile liquid is fixed 3 times, can be at-4 ℃ of long preservation samples.We adopt Ka Nuoshi liquid (methyl alcohol: glacial acetic acid=3: 1, volume ratio) fixing, and 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times.Owing to have glacial acetic acid certain dissociation to be arranged in the immobile liquid to sample, can influence fixed effect, therefore before adding immobile liquid for the first time, only remove half hypotonic medium as buffering, second and third time adds new immobile liquid after then need old liquid being removed fully again.
Dissociate the time: adopt glacial acetic acid to dissociate, concentration is 50%, and the time of dissociating, too short or long all can the influence dripped a sheet effect, and our time set that will dissociate is 45min, and it is respond well to drip sheet, does not have big piece of tissue and disturbs.Remove clean immobile liquid before dissociating, otherwise influence dissociation solution concentration.
Drip the sheet method: cold conventional sheet method repeats to drip sheet easily, influences chromosome observation, and needs dried overnight, and required time is longer.We adopt heat to drip a sheet method to drip sheet, and 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, whenever drip off one after with thieving paper in the drop centre position absorption excess liquid.
Dyeing time: adopt the dyeing of 10%Giemsa dye liquor; Be Giemsa stoste (production of GIBCO company) 1ml, the phosphate buffer of PH 6.8 (PBS) 9ml, dyeing time has finally influenced the quality of chromosome specimen; The too short chromosome morphology of dyeing time is unintelligible; The long background colour of dyeing time is deepened, and the dyeing time that we adopt is 30min, cleans slide with tap water then.
The step of developing a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping; The slide lower end becomes 30 ° with surface level, the slide frosting contacts with current, and current just become wire to drip; Slowly flushing, the water to flushing no longer has till the color.
Adopt said method; We have carried out practical application in Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science; The golden cuttlefish embryo chromosome is carried out film-making, and the result shows, adopts method of the present invention can obtain good chromosome specimen; Chromosome metacinesis is many mutually, form is clear, good dispersion, and concrete effect is seen Fig. 1.
Claims (2)
1. method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen; Its running program is stripping, pre-treatment, hypotonic processing, fixes, dissociates, drips sheet, dyes and develop a film; It is characterized in that described sheet adopts heat to drip the sheet method; 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, whenever drip off one after with thieving paper in the drop centre position absorption excess liquid.
2. method according to claim 1 is characterized in that described stripping: use fixedly ovum of tweezers, needle egg membrane with dissecting needle; Both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo;
Described pre-treatment: the embryo behind the stripping adopts colchicine to carry out pre-treatment, handles 30min in the colchicine solution with embryonic cell immersion 0.04%;
Described hypotonic processing: adopt KCl as hypotonic medium, concentration is 5%, handles 45min, removes clean colchicine before the hypotonic processing;
Described fixing: as to adopt Ka Nuoshi liquid, i.e. methyl alcohol: glacial acetic acid=3: 1 (volume ratio) is fixing, and 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times, need remove half hypotonic medium before adding immobile liquid for the first time;
Described dissociating: adopting concentration is the 50% glacial acetic acid 45min that dissociates, and removes clean immobile liquid before dissociating;
Described sheet: adopt heat to drip the sheet method, 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, whenever drip off one after with thieving paper in the drop centre position absorption excess liquid;
Described dyeing: adopt the dyeing of 10%Giemsa dye liquor, i.e. Giemsa stoste 1ml, the phosphate buffer 9ml of PH 6.8, dyeing time 30min;
Described developing a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping; The slide lower end becomes 30 ° with surface level, the slide frosting contacts with current, and current just become wire to drip; Slowly flushing, the water to flushing no longer has till the color.
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| CN2011102841169A CN102509504B (en) | 2011-09-22 | 2011-09-22 | Method for preparing chromosome specimen with sepia esculenta embryonic cell |
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| CN102509504B CN102509504B (en) | 2013-07-10 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703599A (en) * | 2012-06-26 | 2012-10-03 | 山东东方海洋科技股份有限公司 | Improved preparation method of kelp chromosome |
| CN104483178A (en) * | 2014-11-24 | 2015-04-01 | 中国水产科学研究院黄海水产研究所 | Preparation method of chromosomes of adult epinephelus akaara |
| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
| CN112772554A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Method for improving rate of emergence of giant salamanders in breeding |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4031635A (en) * | 1975-04-14 | 1977-06-28 | Brandt Edward E | Manipulative chromosomal model |
| CN201780226U (en) * | 2010-08-30 | 2011-03-30 | 深圳华大基因科技有限公司 | Chromosome specimen dropping device |
-
2011
- 2011-09-22 CN CN2011102841169A patent/CN102509504B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4031635A (en) * | 1975-04-14 | 1977-06-28 | Brandt Edward E | Manipulative chromosomal model |
| CN201780226U (en) * | 2010-08-30 | 2011-03-30 | 深圳华大基因科技有限公司 | Chromosome specimen dropping device |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703599A (en) * | 2012-06-26 | 2012-10-03 | 山东东方海洋科技股份有限公司 | Improved preparation method of kelp chromosome |
| CN102703599B (en) * | 2012-06-26 | 2014-01-22 | 山东东方海洋科技股份有限公司 | Improved preparation method of kelp chromosome |
| CN104483178A (en) * | 2014-11-24 | 2015-04-01 | 中国水产科学研究院黄海水产研究所 | Preparation method of chromosomes of adult epinephelus akaara |
| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
| CN112772554A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Method for improving rate of emergence of giant salamanders in breeding |
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| CN102509504B (en) | 2013-07-10 |
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