CN104745527A - Novel rapid chick embryonic disc separation method - Google Patents
Novel rapid chick embryonic disc separation method Download PDFInfo
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- CN104745527A CN104745527A CN201510193606.6A CN201510193606A CN104745527A CN 104745527 A CN104745527 A CN 104745527A CN 201510193606 A CN201510193606 A CN 201510193606A CN 104745527 A CN104745527 A CN 104745527A
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- blastodisc
- yolk
- tweezers
- egg
- eggshell
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- 238000000926 separation method Methods 0.000 title claims abstract description 26
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 51
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 51
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 33
- 210000003278 egg shell Anatomy 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 25
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 17
- 210000000969 egg white Anatomy 0.000 claims abstract description 17
- 235000014103 egg white Nutrition 0.000 claims abstract description 17
- 235000013601 eggs Nutrition 0.000 claims abstract description 12
- 210000002298 blastodisc Anatomy 0.000 claims description 65
- 241000287828 Gallus gallus Species 0.000 claims description 32
- 210000001534 vitelline membrane Anatomy 0.000 claims description 15
- 210000001161 mammalian embryo Anatomy 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 230000004720 fertilization Effects 0.000 claims description 5
- 239000011435 rock Substances 0.000 claims description 5
- 235000009161 Espostoa lanata Nutrition 0.000 claims description 4
- 240000001624 Espostoa lanata Species 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 241000519995 Stachys sylvatica Species 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 230000001788 irregular Effects 0.000 claims description 3
- 238000012423 maintenance Methods 0.000 claims description 3
- 230000000877 morphologic effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000005855 radiation Effects 0.000 claims description 2
- 229910001220 stainless steel Inorganic materials 0.000 claims description 2
- 239000010935 stainless steel Substances 0.000 claims description 2
- 238000013022 venting Methods 0.000 claims description 2
- 210000003837 chick embryo Anatomy 0.000 abstract 2
- 239000003814 drug Substances 0.000 description 7
- 230000031787 nutrient reservoir activity Effects 0.000 description 5
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 210000001172 blastoderm Anatomy 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000000998 shell membrane Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
Abstract
The invention discloses a novel rapid chick embryonic disc separation method, which comprises the steps that firstly, the blunt end of a chick embryo is upwards held in a hand, sharp tips of a pair of tweezers gently drill a small hole in the middle of a fertile egg, certain ends of the pair of tweezers are inserted into the small hole, the pair of tweezers is used for clamping an egg shell broken in sequence along the equator line direction, the egg shell is separated for a circle along the equator line, the egg shell of the blunt end is slightly peeled, the chick embryo is kept horizontal as much as possible in the process, and a yolk is prevented from flowing along with egg white; after the egg shell is peeled, the pair of tweezers is used for separating the egg white from the yolk; the yolk is shifted by using the side face of the pair of tweezers to find an embryonic disc; after the embryonic disc is found, the embryonic disc is shifted to the midpoint as much as possible, the embryonic disc is cut off in a square along the periphery of the embryonic disc by a pair of scissors, at the moment, the pair of tweezers is used for clamping one corner of a square yolk film containing the embryonic disc, and gently pulling out along the horizontal direction, the yolk film is placed in a PBS culture dish at the temperature of 37 DEG C, the culture dish is slightly shaken, and the yolk and the yolk film are separated from the embryonic disc, thus obtaining the whole embryonic disc.
Description
Technical field:
The present invention relates to Stem Cell Engineering, the preservation of rareness species and tissue and the field such as cell engineering.
Technical background:
Three kinds of methods are mainly contained to the separation of chicken blastodisc, medicine spoon method of acquiring at present, the around-France and hair ring method of filter paper paper.These three kinds of methods all need, by removing fresh fertile egg blunt end eggshell and shell membrane, to make it become the circular hole of diameter 3 ~ 4cm, expose yolk and blastodisc, and then carry out the separation of blastodisc.Medicine spoon method mainly adopt eye scissors along the edge of blastodisc careful clip blastodisc, medicine spoon is put in the culture dish of the PBS filling 37 DEG C after scooping out.Rock culture dish gently, yolk and vitelline membrane are separated from blastodisc.Then blastodisc carefully takes out by medication spoon from culture dish, puts into the culture dish that another fills warm PBS, carries out follow-up test.Paper is around-France, cuts a filter paper circle, and paper circle, slightly larger than blastodisc, is gently pressed onto below albumen by the diameter of central circular hole, makes it to be close to blastodisc, and makes blastodisc be positioned at circular hole central authorities.Left hand is held ophthalmic tweezers sub-folder and is lived paper circle edge, right handed scissors cuts along paper circle that (blastodisc edge sticks on paper circle, embryo is complete), with tweezers paper circle is moved in the culture dish filling warm PBS, rock gently and the vitelline membrane above blastodisc and yolk below thereof are removed, carry out follow-up test.Hair ring method is cut off by circular blastodisc, chooses in the culture dish filling warm PBS.Reject yolk with hair circular layer layer, then wash several times with PBS, until remove yolk and membrane of yolk completely, carry out follow-up test.
Summary of the invention:
Long for current chicken blastodisc disengaging time, efficiency is low and be separated imperfect, the yolk protein of blastodisc and remain the problems such as more, the present invention adopts and only uses tweezers to be separated the fast separating process of blastodisc, the advantage such as have that velocity of separation is fast, purity is high and blastodisc is complete.Adopt present method, per hour two separable chicken embryos 100 pieces for each person, compare discovery with traditional method, for medicine spoon method conventional at present, the separable chicken embryo of two people per hour 50 pieces, raises the efficiency twice, and the chicken blastodisc be separated is more complete, yolk protein is less, and purity is higher.The separation method of the quick chicken blastodisc that invention proposes has huge application prospect, for the research body of chicken embryonic stem cells provides important technical guarantee.
The object of the invention is as people provide a kind of separation method of quick chicken blastodisc newly.
The present invention includes following steps:
1. the cleaning of chicken embryo
Select fresh fertilization of unhatched X phase kind of egg after being born, using hospital gauze to infiltrate concentration is 0.1% bromogeramine, and after thoroughly cleaning kind of an egg, thorough disinfection is for subsequent use again for the alcohol of use 75%;
2. the preparation of reagent and apparatus;
Secure ph is the 1XPBS damping fluid 1L of 7.5, for subsequent use after high pressure.Prepare 6 90mm culture dish, 2 10cm ophthalmology straight forceps, and 2 14cm scissors straight, 2 16cm suction pipes, two pieces of 50*50cm gauzes, and after cleaning, drying, load stainless steel high-voltage cage mesohigh for subsequent use, cotton ball soaked in alcohol is some;
3. Bechtop is clean
Hospital gauze through 0.1% bromogeramine infiltrate after Bechtop is thoroughly cleaned, re-use after it is air-dry 75% cotton ball soaked in alcohol sterilization, then with wavelength be 250nm-260nm ultraviolet radiation disinfection 30mins rear venting use;
4. operator prepare
Operator wear gown of a doctor, and both hands are 0.1% bromogeramine sterilization through concentration, reuse 75% alcohol disinfecting, after air-dry, wear emgloves and carry out test;
5. the stripping of eggshell
After Bechtop ventilation 15mins, start chicken blastodisc separating experiment, first chicken embryo blunt end is upwards held in the hand, in the middle part of fertile egg, getting out footpath always gently with tweezers tip is 0.2-0.5cm aperture, aperture is inserted in tweezers one end, insert excessively not dark, 0.2-0.5cm is advisable, and along equator direction, is caught broken eggshell successively with tweezers, firmly not too quickly, only be caught broken eggshell in the middle of tweezers, after prolonging equator separation eggshell one circle, stripping blunt end eggshell gently, chicken embryo level should be kept in the process, prevent yolk from flowing out with egg white;
6. the separation of egg white
Peel off after eggshell, remain eggshell part in hand slowly behind inclination 15-20 degree angle, allow egg white naturally flow out, part does not flow out egg white can haul out gently with tweezers, and action need are careful, are sure not yolk to pour out, and the eggshell in most of egg white separation defensive position should recover level;
7. the searching of blastodisc
Use the side of tweezers to stir rotation yolk from the edge of yolk, find fertilization blastodisc, be sure not to use tip;
8. the morphological specificity identification of blastodisc
Chicken oosperm constantly divides in parent, it is the X phase after output, having split into a cell, is that diameter is about 0.5-0.6cm white translucent disk from the surface observation of yolk, and clear egg can only be the irregular small white spots of 0.1-0.2cm to a diameter at yolk surface observation;
9. the separation of blastodisc
After finding blastodisc, with the side of tweezers, blastodisc is pushed centre, be that 1cm*1cm square is cut off with scissors along blastodisc surrounding, the unsuitable tension of distance blastodisc, every side reserves 0.3cm left and right margins, use tweezers clamp by containing blastodisc square vitelline membrane one jiao, pull out gently in the horizontal direction, maintenance level, reduce the yolk quantity of taking out of, above-mentioned vitelline membrane is put into the culture dish of the PBS of 37 DEG C, rock culture dish gently, yolk and vitelline membrane are separated from blastodisc, thus obtains complete blastodisc.
The invention of present method, mainly for improving the separation efficiency of chicken blastodisc, and obtains complete blastodisc, reduces the residual of yolk protein.
Adopt present method, per hour two separable chicken embryos 100 pieces for each person, discovery is compared with traditional method, for medicine spoon method conventional at present, the separable chicken embryo of two people per hour 50 pieces, raise the efficiency twice, greatly shorten the disengaging time of blastodisc, improve separation efficiency, and the chicken blastodisc be separated is more complete, and yolk protein remains less, purity is higher.
Superiority of the present invention is:
1. velocity of separation is fast;
2. method is simple, repeatable strong, can carry out in common lab;
3. the blastodisc using the method to obtain is complete, can meet the needs of ordinary test;
4. adopt present method, per hour 2 separable chicken embryos 100 pieces for each person, find more afterwards with traditional method: for medicine spoon method conventional at present, the separable chicken embryo of 2 people per hour 50 pieces, raise the efficiency 2 times, and the chicken blastodisc be separated is more complete, yolk protein is less, each blastodisc can obtain 5-6 ten thousand embryonic stem cells, and purity is higher;
5., for the separation of avian blastodermal cell provides new method, also provide method and access for chicken blastoderm cell follow-up study from now on simultaneously;
6. this technology is also applicable to the separation of the blastoderm cells of other oviparous animal, can be used for the utilization and exploitation of precious species.
Embodiment
1. the stripping of eggshell
After Bechtop ventilation 15mins, start chicken blastodisc separating experiment, first chicken embryo blunt end is upwards held in the hand, in the middle part of fertile egg, getting out footpath always gently with tweezers tip is 0.2-0.5cm aperture, aperture is inserted in tweezers one end, insert excessively not dark, 0.2-0.5cm is advisable, and along equator direction, is caught broken eggshell successively with tweezers, firmly not too quickly, only be caught broken eggshell in the middle of tweezers, after prolonging equator separation eggshell one circle, stripping blunt end eggshell gently, chicken embryo level should be kept in the process, prevent yolk from flowing out with egg white;
2. the separation of egg white
Peel off after eggshell, remain eggshell part in hand slowly behind inclination 15-20 degree angle, allow egg white naturally flow out, part does not flow out egg white can haul out gently with tweezers, and action need are careful, are sure not yolk to pour out, and the eggshell in most of egg white separation defensive position should recover level;
3. the searching of blastodisc
Use the side of tweezers to stir rotation yolk from the edge of yolk, find fertilization blastodisc, be sure not to use tip;
4. the morphological specificity identification of blastodisc
Chicken oosperm constantly divides in parent, it is the X phase after output, having split into a cell, is that diameter is about 0.5-0.6cm white translucent disk from the surface observation of yolk, and clear egg can only be the irregular small white spots of 0.1-0.2cm to a diameter at yolk surface observation;
5. the separation of blastodisc
After finding blastodisc, with the side of tweezers, blastodisc is pushed centre, be that 1cm*1cm square is cut off with scissors along blastodisc surrounding, the unsuitable tension of distance blastodisc, every side reserves 0.3cm left and right margins, use tweezers clamp by containing blastodisc square vitelline membrane one jiao, pull out gently in the horizontal direction, maintenance level, reduce the yolk quantity of taking out of, above-mentioned vitelline membrane is put into the culture dish of the PBS of 37 DEG C, rock culture dish gently, yolk and vitelline membrane are separated from blastodisc, thus obtains complete blastodisc.
Claims (1)
1. a separation method for new quick chicken blastodisc, is characterized in that: comprise the steps:
(1) cleaning of chicken embryo
Select fresh fertilization of unhatched X phase kind of egg after being born, using hospital gauze to infiltrate concentration is 0.1% bromogeramine, and after thoroughly cleaning kind of an egg, thorough disinfection is for subsequent use again for the alcohol of use 75%;
(2) preparation of reagent and apparatus
Secure ph is the 1XPBS damping fluid 1L of 7.5, for subsequent use after high pressure, and prepare 6 90mm culture dish, 2 10cm ophthalmology straight forceps, 2 14cm scissors straights, 2 16cm suction pipes, two pieces of 50*50cm gauzes, after cleaning, drying, load stainless steel high-voltage cage mesohigh for subsequent use, cotton ball soaked in alcohol is some;
(3) Bechtop is clean
Hospital gauze through 0.1% bromogeramine infiltrate after Bechtop is thoroughly cleaned, re-use after it is air-dry 75% cotton ball soaked in alcohol sterilization, then with wavelength be 250nm-260nm ultraviolet radiation disinfection 30mins rear venting use;
(4) operator prepare
Operator wear gown of a doctor, and both hands are 0.1% bromogeramine sterilization through concentration, reuse 75% alcohol disinfecting, after air-dry, wear emgloves and carry out test;
(5) stripping of eggshell
After Bechtop ventilation 15mins, start chicken blastodisc separating experiment, first chicken embryo blunt end is upwards held in the hand, in the middle part of fertile egg, getting out footpath always gently with tweezers tip is 0.2-0.5cm aperture, aperture is inserted in tweezers one end, insert excessively not dark, 0.2-0.5cm is advisable, and along equator direction, is caught broken eggshell successively with tweezers, firmly not too quickly, only be caught broken eggshell in the middle of tweezers, after prolonging equator separation eggshell one circle, stripping blunt end eggshell gently, chicken embryo level should be kept in the process, prevent yolk from flowing out with egg white;
(6) separation of egg white
Peel off after eggshell, remain eggshell part in hand slowly behind inclination 15-20 degree angle, allow egg white naturally flow out, partly not flowing out egg white can haul out gently with tweezers, is sure not yolk to pour out, and the eggshell in most of egg white separation defensive position should recover level;
(7) searching of blastodisc
Use the side of tweezers to stir rotation yolk from the edge of yolk, find fertilization blastodisc, be sure not to use tip;
(8) the morphological specificity identification of blastodisc;
Chicken oosperm constantly divides in parent, it is the X phase after output, having split into a cell, is that diameter is about 0.5-0.6cm white translucent disk from the surface observation of yolk, and clear egg can only be the irregular small white spots of 0.1-0.2cm to a diameter at yolk surface observation;
(9) separation of blastodisc
After finding blastodisc, with the side of tweezers, blastodisc is pushed centre, be that 1cm*1cm square is cut off with scissors along blastodisc surrounding, the unsuitable tension of distance blastodisc, every side reserves 0.3cm left and right margins, use tweezers clamp by containing blastodisc square vitelline membrane one jiao, pull out gently in the horizontal direction, maintenance level, reduce the yolk quantity of taking out of, above-mentioned vitelline membrane is put into the culture dish of the PBS of 37 DEG C, rock culture dish gently, yolk and vitelline membrane are separated from blastodisc, thus obtains complete blastodisc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510193606.6A CN104745527B (en) | 2015-04-22 | 2015-04-22 | A kind of separation method of new quick chicken embryo disk |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510193606.6A CN104745527B (en) | 2015-04-22 | 2015-04-22 | A kind of separation method of new quick chicken embryo disk |
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| Publication Number | Publication Date |
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| CN104745527A true CN104745527A (en) | 2015-07-01 |
| CN104745527B CN104745527B (en) | 2018-01-05 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286019A (en) * | 2019-04-26 | 2019-09-27 | 青岛农业大学 | A kind of whole embryo fixing means of striping anisolecithal egg |
| CN111676299A (en) * | 2020-07-30 | 2020-09-18 | 扬州大学 | Method for identifying cell types in chick blastocyst paring disc |
| CN112626007A (en) * | 2020-12-15 | 2021-04-09 | 东北农业大学 | Method for extracting early embryo of chicken and method for making atlas |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007035768A2 (en) * | 2005-09-20 | 2007-03-29 | Embrex, Inc. | Methods for rapidly and accurately locating avian egg blastoderms |
| CN101020898A (en) * | 2007-01-12 | 2007-08-22 | 湖南省畜牧兽医研究所 | Process of windowing breeding egg and hatching chicken embryo |
| CN103013909A (en) * | 2012-12-05 | 2013-04-03 | 中国农业大学 | Method of efficiently separating embryonic stem cells of poultry |
-
2015
- 2015-04-22 CN CN201510193606.6A patent/CN104745527B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007035768A2 (en) * | 2005-09-20 | 2007-03-29 | Embrex, Inc. | Methods for rapidly and accurately locating avian egg blastoderms |
| CN101020898A (en) * | 2007-01-12 | 2007-08-22 | 湖南省畜牧兽医研究所 | Process of windowing breeding egg and hatching chicken embryo |
| CN103013909A (en) * | 2012-12-05 | 2013-04-03 | 中国农业大学 | Method of efficiently separating embryonic stem cells of poultry |
Non-Patent Citations (1)
| Title |
|---|
| 张传生: "鸡X期胚盘细胞体外培养于输卵管生物反应器的研制", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286019A (en) * | 2019-04-26 | 2019-09-27 | 青岛农业大学 | A kind of whole embryo fixing means of striping anisolecithal egg |
| CN111676299A (en) * | 2020-07-30 | 2020-09-18 | 扬州大学 | Method for identifying cell types in chick blastocyst paring disc |
| CN112626007A (en) * | 2020-12-15 | 2021-04-09 | 东北农业大学 | Method for extracting early embryo of chicken and method for making atlas |
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| Publication number | Publication date |
|---|---|
| CN104745527B (en) | 2018-01-05 |
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