CN102399821B - Acquisition method of substance of cell nucleus - Google Patents
Acquisition method of substance of cell nucleus Download PDFInfo
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- CN102399821B CN102399821B CN 201010281473 CN201010281473A CN102399821B CN 102399821 B CN102399821 B CN 102399821B CN 201010281473 CN201010281473 CN 201010281473 CN 201010281473 A CN201010281473 A CN 201010281473A CN 102399821 B CN102399821 B CN 102399821B
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Abstract
The invention, belonging to the technical field of biology, relates to an acquisition method of the substance of a cell nucleus for a clone technology, comprising the following steps: (1) perforating on the zona pellucid portion with large perivitelline space by laser; (2) fixing the cell through an ovum holding pin to let the open end of the zona pellucid be opposite to the ovum holding pin; (3)pushing an injection needle with liquid into cytoplasm from the opening of the zona pellucid and avoiding the direct contact with the substance of the cell nucleus; (4) rapidly increasing positive pressure in the injection needle to allow the liquid in the injection needle rapidly flow outwards to push and cut the connection between the substance of the cell nucleus and the cytoplasm to make no cytoplasm around the substance of the cell nucleus, and using the increasement of the positive pressure in the whole cell to allow the substance of the cell nucleus completely to be removed from the cell from the opening of the zona pellucid; and (5) using the injection needle to collect the separated substance of the cell nucleus. The method has no need of special devices, can realize the separation of the substance of the cell nucleus simply and conveniently, and can avoid the facture of the substance of the cell nucleus, and the obtained substance of the cell nucleus is convenient for cell fusion experiments.
Description
Technical field
The invention belongs to biological technical field, relate to the method for transitional cell genetic material, relate more specifically to a kind of method of obtaining nuclear material.
Background technology
Nuclear material is determining biological hereditary property as genetic material, and nucleus is the main gathering place of genetic material.Pronuclear-stage embryos has the coated clear protokaryon of complete nuclear membrane, can be in the situation that observe accurately with inverted microscope without any need for utility appliance and reagent, and this is for obtaining accurately and the transfer material provides necessary precondition.Protokaryon obtain manner used is mainly biopsy needle and draws the kytoplasm method at present, namely with near the kytoplasm biopsy needle absorption protokaryon, finally makes the related parts of fine after birth of protokaryon and tenuigenin form a new nucleoid.But there are some following shortcomings in the method: must merge or other amalgamation modes by means of electricity when 1, new nucleoid being transferred to new recipient cytoplasm body; 2, protokaryon easily is subject to the extruding of kytoplasm and breaks in suction process, and this is unacceptable in clinical application in the future; 3, inevitably bring the parts of fine kytoplasm into.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of convenient, fast and method of accurately obtaining nuclear material.
The method that nucleus of the present invention obtains comprises following steps:
(1) laser that is 1.0-1.8ms with frequency is partly beaten the hole of diameter 12-30 μ m at the larger zona pellucida of cell ovum week gap;
(2) fix cell with the ovum pin of holding of external diameter 100～120 μ m, internal diameter 15～30 μ m, make the zona pellucida opening end be positioned at the position relative with holding the ovum pin, conveniently to carry out subsequent operations;
(3) penetrate endochylema from the zona pellucida opening part with the entry needle of inhaling in advance certain quantity of fluid, internal diameter 8-50 μ m, and to avoid directly touching nuclear substance, described amount of liquid can be 0.05 μ l ~ 1 μ l;
(4) increase sharply malleation in entry needle makes the outside rapid flow of liquid in pin, to thrust being connected between nuclear substance and endochylema, make nuclear substance on every side without any kytoplasm part, and utilize the raising of malleation in whole cell to make complete being pressed from cell from the zona pellucida opening part of nuclear substance;
(5) collect with entry needle the nuclear substance that separates.
Wherein, described cell can be ovocyte.
Wherein, the liquid that the liquid described in step (3)～(4) can ooze with various cell culture fluids, operation liquid or other and cell etc. as mHTF, GMOPS, G-IVF or G1.5 nutrient solution etc., preferably contains 5%HSA in described liquid.
Wherein, the nuclear substance described in step (3)～(5) can be GV bubble or other intracellular organic matter that is coated with by complete film of GV phase ovocyte.
Wherein, the preferred 10 μ m of the internal diameter of step (3)～(5) described entry needle.
Wherein, the described ovum pin opening of holding of step (2) can be angle or flat mouth, preferably passes through top-notch angle.
Utilize method of the present invention to carry out nuclear transplantation and compare with current micrurgy method of nuclear transfer, have obvious superiority:
(1) do not need special equipment, only need general inverted microscope and micromanipulation instrument just can carry out, easy and simple to handle, be convenient to extensively promote the use of;
(2) can be with complete the separating with endochylema of nuclear substance;
(3) can not break because extruding makes nuclear substance in the nuclear substance transfer process;
(4) nuclear substance that obtains is without any tenuigenin and cytolemma.
Description of drawings
Fig. 1: partly punch at the larger zona pellucida of ovocyte ovum week gap with laser, and make the zona pellucida opening end be positioned at the position relative with holding the ovum pin; Wherein, 1 is the hole that laser is beaten.
Fig. 2: entry needle penetrates endochylema from the zona pellucida opening part, and avoids directly touching nuclear substance; Wherein, the 2nd, entry needle, the 3rd, nuclear substance.
Fig. 3: what nuclear substance was complete is pressed from ovocyte; Wherein, the 4th, nuclear substance.
Fig. 4: collect the nuclear substance that separates with entry needle; Wherein, the 5th, nuclear substance.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
Embodiment
(1) partly beat the hole of diameter 30 μ m at the larger zona pellucida of GV phase ovocyte ovum week gap with laser, laser frequency is 1.8ms;
(2) fix ovocyte with the ovum pin of holding of external diameter 120 μ m, internal diameter 25 μ m, make the zona pellucida opening end be positioned at the position relative with holding the ovum pin, wherein holding ovum pin opening is flat mouth;
(3) with suction has 1 μ l to contain the G1.5 nutrient solution of 5%HSA in advance, the entry needle of internal diameter 40 μ m penetrates endochylema from the zona pellucida opening part, and avoid directly touching the GV bubble;
(4) increase sharply malleation in entry needle makes the outside rapid flow of liquid in pin, makes the liquid of entry needle thrust fast being connected between GV bubble and endochylema, and utilizes the raising of malleation in whole cell to make GV finish whole being pressed from ovocyte;
(5) collect with entry needle the GV bubble that separates.
Claims (9)
1. the acquisition methods of a nuclear material comprises the following steps:
(1) partly beat the hole of diameter 12-30 μ m at the larger zona pellucida of cell ovum week gap with laser;
(2) fix cell with the ovum pin of holding of external diameter 100～120 μ m, internal diameter 15～30 μ m, make the zona pellucida opening end be positioned at the position relative with holding the ovum pin;
(3) there is the entry needle of 0.05 μ l ~ 1 μ l liquid, internal diameter 8-50 μ m to penetrate endochylema from the zona pellucida opening part with inhaling, and avoids directly touching nuclear substance;
(4) increase sharply malleation in entry needle makes the outside rapid flow of liquid in pin, to thrust being connected between nuclear substance and endochylema, make nuclear substance on every side without any kytoplasm part, and utilize the raising of malleation in whole cell to make complete being pressed from cell from the zona pellucida opening part of nuclear substance;
(5) collect with entry needle the nuclear substance that separates;
Wherein said cell is ovocyte.
2. method according to claim 1, is characterized in that, described nuclear substance is GV bubble or other intracellular organic matter that is coated with by complete film of GV phase ovocyte.
3. method according to claim 1, is characterized in that, the liquid described in step (3)～(4) is the liquid that various cell culture fluids, operation liquid or other and cell etc. ooze.
4. method according to claim 3, is characterized in that, described liquid is mHTF, GMOPS, G-IVF or G1.5 nutrient solution.
5. method according to claim 4, is characterized in that, contains 5%HSA in described liquid.
6. method according to claim 1, is characterized in that, the internal diameter of step (3)～(5) described entry needle is 10 μ m.
7. method according to claim 1, is characterized in that, the described ovum pin opening of holding of step (2) is angle or flat mouth.
8. method according to claim 7, is characterized in that, the described ovum pin opening of holding is the top-notch angle of process.
9. method according to claim 1, is characterized in that, laser frequency is 1.0 ~ 1.8ms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010281473 CN102399821B (en) | 2010-09-13 | 2010-09-13 | Acquisition method of substance of cell nucleus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010281473 CN102399821B (en) | 2010-09-13 | 2010-09-13 | Acquisition method of substance of cell nucleus |
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| CN102399821A CN102399821A (en) | 2012-04-04 |
| CN102399821B true CN102399821B (en) | 2013-05-22 |
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| CN 201010281473 Active CN102399821B (en) | 2010-09-13 | 2010-09-13 | Acquisition method of substance of cell nucleus |
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| CN109735487A (en) * | 2018-12-18 | 2019-05-10 | 陈子江 | A kind of constraint method of lensless Fourier transform holography cell |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769442A (en) * | 2005-10-13 | 2006-05-10 | 莱阳农学院 | Method for removing mammal ovocyte karyon |
| CN1807616A (en) * | 2006-01-13 | 2006-07-26 | 山东大学 | Transplantation method for nucleus |
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- 2010-09-13 CN CN 201010281473 patent/CN102399821B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769442A (en) * | 2005-10-13 | 2006-05-10 | 莱阳农学院 | Method for removing mammal ovocyte karyon |
| CN1807616A (en) * | 2006-01-13 | 2006-07-26 | 山东大学 | Transplantation method for nucleus |
Non-Patent Citations (4)
| Title |
|---|
| Human Embryos Derived by Somatic Cell Nuclear Transfer Using an Alternative Enucleation Approach;Jianyuan Li等;《Cloning and Stem Cells》;20090312;第11卷(第1期);39-50 * |
| Jianyuan Li等.Human Embryos Derived by Somatic Cell Nuclear Transfer Using an Alternative Enucleation Approach.《Cloning and Stem Cells》.2009,第11卷(第1期),39-50. |
| 吴克良等.用改进的细胞核移植方法构建重构胚.《生物工程学报》.2007,第23卷(第01期),161-165. |
| 用改进的细胞核移植方法构建重构胚;吴克良等;《生物工程学报》;20070130;第23卷(第01期);161-165 * |
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