CN102876719A - Electro-fusion method in somatic cell nuclear transfer - Google Patents
Electro-fusion method in somatic cell nuclear transfer Download PDFInfo
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- CN102876719A CN102876719A CN2012104197058A CN201210419705A CN102876719A CN 102876719 A CN102876719 A CN 102876719A CN 2012104197058 A CN2012104197058 A CN 2012104197058A CN 201210419705 A CN201210419705 A CN 201210419705A CN 102876719 A CN102876719 A CN 102876719A
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Abstract
The invention discloses an electro-fusion method in somatic cell nuclear transfer. The electro-fusion method comprises the following steps: S1. transferring recovered reconstructed oocytes into a fusion solution in batches for balancing; S2. washing the reconstructed oocytes by using a fusion activating solution; S3. putting the washed reconstructed oocytes into a fusion groove filled with the fusion solution, adjusting a membrane contact surface of donor cells-receptor oocytes to be parallel to an electrode, enabling one end of each donor cell to face to the negative pole of the electrode, and applying direct-current pulse by using a fusion instrument to induce the fusion and simultaneously activate the reconstructed oocytes; and S4. washing the fused and activated reconstructed oocytes by using an embryo culture solution, then transferring the reconstructed oocytes into a culture box, and fusing the reconstructed oocytes in the embryo culture solution covered by mineral oil or an auxiliary activating solution. According to the method, in the process of fusion, one end of each donor cell faces to the negative pole of the electrode, so that the fusion efficiency is improved.
Description
Technical field
The present invention relates to the nucleus fusion method, be specifically related to the electric fusion method in a kind of body-cell neucleus transplanting.
Background technology
The animal cloning technology that nuclear transplantation (nuelear transfer or nuelear transplantation) technology also namely is widely known by the people.Normally by micrurgic method, the nucleus of donorcells is injected in the mature oocyte or early stage zygote of in advance stoning, form a new caryoplasm recombinant chou (being the reconstruct ovum), the nuclear that moves into is under the effect of recipient cytoplasm, genesis and development program rearrange (Reprogramming), so that the caryoplasm recombinant chou is the same with the normal fertilization ovum, through cell fission, differentiation and in parent, develop into a new individuality (Zhang Yunhai, 2005), the individuality of its acquisition has identical inherited character with the parent.
The nucleus of donorcells is imported in the ovocyte matter, and this process is referred to as to merge.The fusion method of using the earliest is to use Sendai virus (HVJ) (McGrath and Solter, the 1983) mediated cell of deactivation to merge, and obtained success, but effect is very unstable and toxicity is large.What Willadsen etc. (1986) adopted in utilizing the test of embryonic cell clone sheep is the method for direct current mediates fusion, easy and efficient, electro fusion also begins to grow up from this time, its principle is the cell membrane phospholipid bilayer recurring structure change that makes close contact by the stimulation of electric current, and then form intercellular duct, along with enlarging gradually, the duct makes the kytoplasm blending together.Become at present the fusion method that generally adopts in the animal cloning.
Yet, because the impact of the factors such as the difference in the technological operation and donorcells type causes fusion rate unstable, be the important factor that affects cloning efficiency.In the prior art general operation is: after the reconstruct ovum is put into integration slot, namely apply after making donorcells-recipient oocyte film contact surface and electrode being parallel different parameters electric pulse induce fusion, and do not consider somatocyte towards the impact of negative or positive electrode on fusion efficiencies, thereby make fusion efficiencies very unstable.
Summary of the invention
Goal of the invention of the present invention is to overcome the defective of prior art, provides a kind of fusion efficiencies high electric fusion method.
For achieving the above object, the present invention adopts following technical scheme, and the electric fusion method in a kind of body-cell neucleus transplanting may further comprise the steps:
S1, the reconstruct ovum after will recovering are transferred in batches and are merged balance in the liquid;
S2, usefulness merge activation solution washing reconstruct ovum;
S3, the reconstruct ovum after will washing are put into the integration slot that merges liquid are housed, adjustment donorcells-recipient oocyte film contact surface is parallel with electrode and make donorcells one end towards the negative pole of electrode, applies electric pulse with fusion instrument and induces fusion to activate simultaneously the reconstruct ovum;
S4, the reconstruct ovum that will merge after activating wash with embryo medium, then change in the incubator, in the embryo medium that mineral oil covers or merge in the assisted activation liquid;
The method with the negative pole of donorcells one end towards electrode, has improved fusion efficiencies in the process that merges.
Containing concentration in the described fusion activation solution is that 0.2 ∽ 0.5mmol/L N.F,USP MANNITOL, concentration are 0.05 ∽ 0.2mmol/L CaCl
22H
2O, concentration are 0.05 ∽ 0.2mmol/L MgCl
26H
2O, concentration are that 0.5 ∽ 2mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid, concentration are 0.01% polyvinyl alcohol (W/V).
The time of balance is 1-5 minute among the described step S1.
Every batch of quantity of putting into the reconstruct ovum that the integration slot that merges liquid is housed is 5-8 among the described step S1.
Merge activation parameter among the described step S3: strength of electric field is 80-100v/mm, and the burst length is 80-100 μ s, implements electricimpulse 2-3 time.
Temperature among the described step S4 in the incubator is 38.5 ℃ or 39 ℃.
The saturation ratio of CO2 among the described step S4 in the incubator is 5%.
Beneficial effect of the present invention adopts this fusion method can significantly improve fusion efficiencies.
Embodiment
The nucleus of donorcells is injected into non-nucleus egg mother cell forms later on the reconstruct ovum, because this process is the process of an injury to ovocyte, need be positioned in the incubator, after the ovocyte kytoplasm recovers 30min, be beneficial to the mixing operation of back, i.e. recovery process.
Every batch of reconstruct ovum after 5-8 recovered is transferred to and is merged balance 2min in the liquid, when changing a kind of liquid, all need put in advance corresponding liquid, is equivalent to the process of an adaptation, i.e. balance.
With merging activation solution washing 3 times, reconstruct ovum after will washing is again put into and is paved with the integration slot that merges liquid, stir recombinant eggs with the very thin solid glass pin that draws, make donorcells-recipient oocyte film contact surface simultaneously make donorcells one end parallel with electrode all towards the negative pole of electrode, with BLS-150B fusion instrument (Hungary) apply different fusion parameters electric pulse induce fusion to activate simultaneously, wherein merging activation parameter is: strength of electric field is 80-100v/mm, electrical pulse time is 80-100 μ s, implements electricimpulse 2-3 time.Then the reconstruct ovum is washed 3 times with nutrient solution, reconstruct ovum after will washing immediately changes in the embryo medium that mineral oil covers or in the assisted activation liquid, be placed into 38.5 ℃ or 39 ℃, cultivate in the incubator of 5%CO2 saturation ratio, behind the 4h under stereoscopic microscope decision fusion.Wherein merge contain the N.F,USP MANNITOL that concentration is 0.2 ∽ 0.5mmol/L (Mannitol) in the activation solution, concentration is the CaCl of 0.05 ∽ 0.2mmol/L
22H
2O, concentration are the Mg Cl of 0.05 ∽ 0.2mmol/L
26H
2O, concentration are that 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), the concentration of 0.5 ∽ 2mmol/L is 0.01%(W/V) polyvinyl alcohol (PVA).Through test of many times, obtain the result such as table 1:
Somatocyte is towards the impact on nuclear transplantation efficient when table 1, fusion
Experiment repeats 11 times, fusion rate significant difference (P=0.006＜0.05), and spilting of an egg rate difference is (P=0.59〉0.05) not significantly, and blastaea rate difference is (P=0.27〉0.05) not significantly
As can be seen from Table 1, when somatocyte during towards negative pole, the efficient of fusion does not affect again spilting of an egg rate and blastaea rate apparently higher than somatocyte simultaneously towards the efficient of positive pole.
Claims (7)
1. the electric fusion method in the body-cell neucleus transplanting is characterized in that, may further comprise the steps:
S1, the reconstruct ovum that will recover to get well are transferred in batches and are merged balance in the activation solution;
S2, usefulness merge activation solution washing reconstruct ovum;
S3, the reconstruct ovum after will washing are put into the integration slot that merges activation solution are housed, adjust donorcells-recipient oocyte film contact surface and make it parallel with electrode and make donorcells one end all towards the negative pole of electrode, apply electric pulse with fusion instrument and induce fusion to activate simultaneously the reconstruct ovum;
S4, the reconstruct ovum that will merge after activating wash with embryo medium, then change in the incubator, in the embryo medium that mineral oil covers or merge in the assisted activation liquid.
2. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, containing concentration in the described fusion activation solution is that 0.2 ∽ 0.5mmol/L N.F,USP MANNITOL, concentration are 0.05 ∽ 0.2mmol/L CaCl
22H
2O, concentration are 0.05 ∽ 0.2mmol/L MgCl
26H
2O, concentration are that 0.5 ∽ 2mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid, concentration are 0.01% polyvinyl alcohol (W/V).
3. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, the time of balance is 1-5 minute among the described step S1.
4. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, every batch of quantity that moves into the reconstruct ovum that merges the liquid balance is 5-8 among the described step S1.
5. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, merges activation parameter among the described step S3: strength of electric field is 80-100v/mm, and the burst length is 80-100 μ s, implements electricimpulse 2-3 time.
6. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, the temperature among the described step S4 in the incubator is 38.5 ℃ or 39 ℃.
7. the electric fusion method in the body-cell neucleus transplanting according to claim 1 is characterized in that, the saturation ratio of the CO2 among the described step S4 in the incubator is 5%.
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| CN2012104197058A CN102876719A (en) | 2012-10-27 | 2012-10-27 | Electro-fusion method in somatic cell nuclear transfer |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039305A (en) * | 2015-07-06 | 2015-11-11 | 广东温氏食品集团股份有限公司 | Improved method used for cloned embryo construction |
| US9249385B1 (en) | 2014-12-18 | 2016-02-02 | City University Of Hong Kong | System and method for fusing cells |
| CN107529743A (en) * | 2015-04-02 | 2018-01-02 | 格尼亚Ip控股私人有限公司 | The processing of biological sample |
Citations (2)
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|---|---|---|---|---|
| CN1733909A (en) * | 2005-07-06 | 2006-02-15 | 广西大学 | Highly effective merge method for animal somatic nucleus transplantation |
| CN1834256A (en) * | 2006-04-03 | 2006-09-20 | 西北农林科技大学 | Needle-shaped microelectrode for nucleus transplantation/clone electrofusion |
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2012
- 2012-10-27 CN CN2012104197058A patent/CN102876719A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733909A (en) * | 2005-07-06 | 2006-02-15 | 广西大学 | Highly effective merge method for animal somatic nucleus transplantation |
| CN1834256A (en) * | 2006-04-03 | 2006-09-20 | 西北农林科技大学 | Needle-shaped microelectrode for nucleus transplantation/clone electrofusion |
Non-Patent Citations (2)
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| 任子利等: "猪手工体细胞核移植电融合／激活参数的优化", 《畜牧兽医学报》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9249385B1 (en) | 2014-12-18 | 2016-02-02 | City University Of Hong Kong | System and method for fusing cells |
| CN107529743A (en) * | 2015-04-02 | 2018-01-02 | 格尼亚Ip控股私人有限公司 | The processing of biological sample |
| CN105039305A (en) * | 2015-07-06 | 2015-11-11 | 广东温氏食品集团股份有限公司 | Improved method used for cloned embryo construction |
| CN105039305B (en) * | 2015-07-06 | 2018-04-10 | 广东温氏食品集团股份有限公司 | A kind of improved method of clone embryos structure |
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