CN102399821A - Acquisition method of substance of cell nucleus for clone technology - Google Patents
Acquisition method of substance of cell nucleus for clone technology Download PDFInfo
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- CN102399821A CN102399821A CN2010102814735A CN201010281473A CN102399821A CN 102399821 A CN102399821 A CN 102399821A CN 2010102814735 A CN2010102814735 A CN 2010102814735A CN 201010281473 A CN201010281473 A CN 201010281473A CN 102399821 A CN102399821 A CN 102399821A
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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 clear protokaryon that complete nuclear membrane encapsulates, and can under the situation of utility appliance and reagent, observe accurately with inverted microscope, and transfer material provides the necessary precondition condition in order to obtain also accurately for this.As treatment offspring's because plasma inheritance (mainly being mitochondrial inheritance material defective) causes securing good health potential means, protokaryon shifts (pronuclear transfer) and is just receiving increasing attention.Used protokaryon obtain manner is mainly biopsy needle and draws the kytoplasm method at present, promptly with near the kytoplasm the biopsy needle absorption protokaryon, finally makes related parts of fine after birth of protokaryon and tenuigenin form a new nucleoid.But there are some following shortcomings in this method: must merge or other amalgamation modes by means of electricity when 1, new nucleoid being transferred to new recipient cytoplasm body; 2, protokaryon and easily in suction process, receive the extruding of kytoplasm and break, this is unacceptable in clinical application in the future; 3, inevitably bring part donorcells matter into, in new embryo's leading role in mid-term of forming, will lose the meaning that protokaryon shifts so like this part donor deficient cells matter.
Summary of the invention
To the deficiency of above-mentioned 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) use frequency as the laser of 1.0-1.8ms in cell ovum week crack bigger zona pellucida partly beat the hole of diameter 12-30 μ m;
(2) fix cell with the ovum pin of holding of external diameter 100～120 μ m, internal diameter 15～30 μ m, the zona pellucida opening end is positioned at and hold the relative position of ovum pin, conveniently to carry out subsequent operations;
(3) penetrate the 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, said amount of liquid can be 0.05 μ l～1 μ l;
(4) malleation that increases sharply in the entry needle outwards flows the liquid in the pin fast; To thrust being connected between nuclear substance and the endochylema; Making does not have any kytoplasm part around the nuclear substance, and utilizes the raising of malleation in the whole cell to make complete being pressed from cell from the zona pellucida opening part of nuclear substance;
(5) collect isolating nuclear substance with entry needle.
Wherein, said cell can be fertilized egg cell or ovocyte.
Wherein, the liquid described in step (3)～(4) can be used various cell culture fluids, operation liquid or other and the isoosmotic liquid of cell, like mHTF, GMOPS, G-IVF or G1.5 nutrient solution or the like, contains 5%HSA in the preferably said liquid.
Wherein, the nuclear substance described in step (3)～(5) can be GV bubble or other intracellular organic matter that is encapsulated by complete film of protokaryon, 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, have obvious superiority with current micrurgy nuclear transplantation method:
(1) do not need special devices, only need general inverted microscope and micromanipulation appearance just can carry out, easy and simple to handle, be convenient to extensively promote the use of;
(2) can be with complete the separating of nuclear substance with endochylema;
(3) can not break in the nuclear substance transfer process owing to extruding makes nuclear substance;
(4) nuclear substance that is obtained does not have any tenuigenin and cytolemma;
(5) in the process that is transferred to recipient cytoplasm once more of nuclear substance, need not pass through electric fusion steps.
Description of drawings
Fig. 1: bigger zona pellucida partly punches in embryo or ovocyte ovum week crack with laser, and the zona pellucida opening end is positioned at and holds the relative position of ovum pin; Wherein, 1 is the hole that laser is beaten.
Fig. 2: entry needle penetrates the 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 isolating nuclear substance with entry needle; Wherein, the 5th, nuclear substance.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described further:
Embodiment 1
(1) with laser in zygote week protokaryon phase crack bigger zona pellucida partly beat the hole of diameter 20 μ m, laser frequency is 1.0ms;
(2) as shown in Figure 1, with external diameter 120 μ m, the ovum pin of holding of internal diameter 25 μ m is fixed zygote, and the zona pellucida opening end is positioned at and hold the relative position of ovum pin, and wherein holding ovum pin opening is angle;
(3) as shown in Figure 2, with suction has 0.5 μ l to contain the GMPOS manipulation in vitro liquid of 5%HSA in advance, the entry needle of internal diameter 10 μ m penetrates the endochylema from the zona pellucida opening part, and avoids directly touching protokaryon;
(4) as shown in Figure 3; The malleation that increases sharply in the entry needle outwards flows the liquid in the pin fast; Make the liquid of entry needle thrust being connected between protokaryon and the endochylema fast, and utilize the raising of malleation in the whole cell to make complete being pressed of protokaryon from zygote;
(5) as shown in Figure 4, collect isolating protokaryon with entry needle.
Embodiment 2
(1) with laser in GV phase ovocyte ovum week crack bigger zona pellucida partly beat the hole of diameter 30 μ m, 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, the zona pellucida opening end is positioned at and hold the relative position of ovum pin, wherein hold ovum pin opening and be flat mouthful;
(3), and avoid directly touching the GV bubble 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 the endochylema from the zona pellucida opening part;
(4) malleation that increases sharply in the entry needle outwards flows the liquid in the pin fast, makes the liquid of entry needle thrust being connected between GV bubble and the endochylema fast, and utilizes the raising of malleation in the whole cell to make GV finish whole being pressed from ovocyte;
(5) collect isolating GV bubble with entry needle.
Claims (10)
1. the acquisition methods of a nuclear material may further comprise the steps:
(1) with laser in cell ovum week crack bigger zona pellucida partly beat the hole of diameter 12-30 μ m;
(2) fix cell with the ovum pin of holding of external diameter 100～120 μ m, internal diameter 15～30 μ m, the zona pellucida opening end is positioned at and holds the relative position of 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 the endochylema with inhaling, and avoids directly touching nuclear substance from the zona pellucida opening part;
(4) malleation that increases sharply in the entry needle outwards flows the liquid in the pin fast; To thrust being connected between nuclear substance and the endochylema; Making does not have any kytoplasm part around the nuclear substance, and utilizes the raising of malleation in the whole cell to make complete being pressed from cell from the zona pellucida opening part of nuclear substance;
(5) collect isolating nuclear substance with entry needle.
2. method according to claim 1 is characterized in that, said cell is fertilized egg cell or ovocyte.
3. method according to claim 1 is characterized in that, said nuclear substance is GV bubble or other intracellular organic matter that is encapsulated by complete film of protokaryon, GV phase ovocyte.
4. method according to claim 1 is characterized in that, the liquid described in step (3)～(4) is various cell culture fluids, operation liquid or other and the isoosmotic liquid of cell.
5. method according to claim 4 is characterized in that, said liquid is mHTF, GMOPS, G-IVF or G1.5 nutrient solution.
6. method according to claim 5 is characterized in that, contains 5%HSA in the said liquid.
7. method according to claim 1 is characterized in that, the internal diameter of step (3)～(5) described entry needle is 10 μ m.
8. method according to claim 1 is characterized in that, the described ovum pin opening of holding of step (2) is angle or flat mouth.
9. method according to claim 8 is characterized in that, the said ovum pin opening of holding is the top-notch angle of process.
10. 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 |
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| 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 true CN102399821A (en) | 2012-04-04 |
| CN102399821B 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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
- 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 (2)
| Title |
|---|
| JIANYUAN LI等: "Human Embryos Derived by Somatic Cell Nuclear Transfer Using an Alternative Enucleation Approach", 《CLONING AND STEM CELLS》 * |
| 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》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735487A (en) * | 2018-12-18 | 2019-05-10 | 陈子江 | A kind of constraint method of lensless Fourier transform holography cell |
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| CN102399821B (en) | 2013-05-22 |
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