CN102703511A - Fuse method for animal body nucleus transfer - Google Patents
Fuse method for animal body nucleus transfer Download PDFInfo
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- CN102703511A CN102703511A CN2012101944035A CN201210194403A CN102703511A CN 102703511 A CN102703511 A CN 102703511A CN 2012101944035 A CN2012101944035 A CN 2012101944035A CN 201210194403 A CN201210194403 A CN 201210194403A CN 102703511 A CN102703511 A CN 102703511A
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Abstract
The invention discloses a fusion method for animal body nucleus transfer. The fusion method comprises the following steps: firstly, placing two half-egg cells into an operation liquid of which the concentration of phytolectin is 0.5-5 mg/ml and bonding to form an egg-egg cell pair; secondly, placing the bonded egg-egg cell pair into a fusion liquid and balancing for 1-3 minutes; thirdly, placing the balanced egg-egg cell pair into an electrofusion tank for electrofusion and applying an alternating current field for instantly positioning the egg-egg cell pair; fourthly, transferring the fused egg-egg cell pair into an operation liquid containing a donor cell to be bonded with the donor cell to form a body-egg cell; and fifthly, transferring the bonded body-egg cell into a fusion excitation liquid and balancing for 1-3 minutes and transferring the balanced body-egg cell into a fusion cell for electrofusion and simultaneously activating. According to the method adopted by the invention, two half-egg cells are fused and then the fused egg-egg cell is fused with the donor cell, and thus the contacted area between the egg cell and the donor cell is increased and simpler operation is realized.
Description
Technical field
The present invention relates to the animal cloning technical field, be specifically related to a kind of fusion method of cloning animal somatic cell nuclear transplantation in the process.
Background technology
1997; The first somatic cell clone sheep in the world " many jasmines (Dolly) (Wilmut et al; 1997) " is born; For the first time negated " the genome experience is irreversible in somatic cell nuclear " this traditional concept in the world, disclosed well differentiated somatic cell nuclear ability reprogramming (Reprogramming) in stoning (removing karyomit(e)) ooecium matter, the declaration mankind have got into the New Times that the higher animal somatocyte can carry out vegetative propagation (clone); Cause a sensation in international community, somatic cell clone becomes the focus of a whole world scientist research thus.Animal cloning is meant that animal directly obtains to have identical inherited character offspring's process with the parent without amphigenetic mode, comprises that parthenogenesis, blastomere separation and cultivation, embryo are cut apart and nuclear transplantation etc. (Sang Runzi, 2002).The method that produces cloned animal then is referred to as the animal cloning technology.At present, in fact the animal cloning technology that is widely known by the people is exactly the phalangeal cell nuclear transfer technology.Nuclear transplantation (nuelear transfer or nuelear transplantation) typically refers to through micrurgic method; The nucleus of donorcells is injected in the mature oocyte or early stage zygote of stoning in advance; Form a new caryoplasm recombinant chou; The nuclear that moves into is under the effect of recipient cytoplasm, and the genesis and development program is rearranged (Reprogramming), makes the caryoplasm recombinant chou the same with the normal fertilization ovum; Through cell fission, differentiation and in parent, develop into a new individuality (Zhang Yunhai, 2005).
(handmade cloning, HMC) technology is a clone technology that does not need the operation of micrurgy appearance to manual clone.Under stereoscopic microscope, directly cut stoning with special cutter, then with two do not contain nucleolate half ovum and donorcells near and impose electric pulse and make its fusion, at last with the single cultivation of reconstructed embryo.Because this method need be removed zona pellucida, and with two and half ooecium plastids and donorcells fusion, thereby also be called as the two half ovum methods (Peura, 1998) of zona pellucida.Again because the whole process of this method leans on manual operation can accomplish and not need some accurate instruments, Vajta etc. (Vajta et al, 2001) are referred to as manual clone with it simultaneously.
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 has obtained success, but very instability and toxicity are big for effect.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; The electricity integration technology begins to grow up also from this time; Its principle is to make the cell membrane phospholipid bilayer recurring structure change of tight contact through the stimulation of electric current, and then forms intercellular duct, makes the kytoplasm blending together along with the duct enlarges gradually.Become at present the fusion method that generally adopts in the animal cloning.
Yet, little because the volume of donorcells is less with the contact area of ovocyte, can influence the fusion rate of donorcells and ovocyte.Particularly in manual clone operations; Ovocyte generally adopts the patterning method stoning, can remove about 1/3 kytoplasm, because the excision kytoplasm is too much; What adopt is that two half ovum merge; Promptly elder generation is bonding with one and half ovum and one piece of donorcells, puts into back, electric integration slot alternating-electric field location again and makes somatocyte align the back fusion through manual stirring, and half ovum after merging then merges with another piece half ovum again.In actual mechanical process, have several problems: 1, ovocyte diminishes also can influence operation, thus the fusion of influence and donorcells; 2, doing when once merging is to lean on to be affixed on the integration slot one rear flank somatocyte that sets right again behind the alternating-current location, in the operation half ovum is dispersed; 3, this fusion method merges donorcells earlier, and operation is trouble relatively, disperses if declare when merging ovum afterwards, can waste bigger workload.
Summary of the invention
Goal of the invention of the present invention is to overcome the defective of prior art, and a kind of fusion method of simple to operate, animal somatic cell nuclear transplantation that syncretizing effect is good is provided.
For realizing that the present invention of foregoing invention purpose adopts following technical scheme, the fusion method in a kind of animal somatic cell nuclear transplantation may further comprise the steps:
(1), two and half ovum being put into phytohemagglutinin concentration is that the bonding formation ovum-ovum of operation liquid of 0.5-5mg/ml is right;
(2), bonding good ovum-ovum was merged the liquid balance 1-3 minute to putting into;
(3), the ovum-ovum after the balance is merged putting into electric integration slot electricity, and apply alternating-current field to ovum-ovum to carrying out the moment location;
(4), will merge in good ovum-ovum contains donorcells to immigration the operation liquid and the bonding organizer-ovum of donorcells;
(5), bonding good body-ovum moved into merged in the exciting liquid balance 1-3 minute, be moved into again and carry out electricity in the integration slot and merge activation simultaneously.
The method that the present invention adopted merges two and half ovum earlier, and ovum-ovum after will merging again and donorcells merge, and increases the contact area of ovum and donorcells, and is simpler in the operation.
The present invention further improves and is, step (1) is preceding puts into the operation liquid 3-8 second of containing phytohemagglutinin respectively with said two and half ovum earlier carrying out.
Optimal way of the present invention merges described in the step (2) in the liquid, and the 4-HEPES and the concentration that contain N.F,USP MANNITOL, concentration that concentration is 0.2 ∽ 0.5mmol/L and be 0.5 ∽ 2mmol/L are the Z 150PH of 0.01g/L.
Optimal way of the present invention, merge in the exciting liquid described in the step (5): containing concentration 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 Mg Cl
26H
2O, concentration are that 0.5 ∽ 2mmol/L 4-HEPES, concentration are the 0.01g/L Z 150PH.
Beneficial effect of the present invention:
Earlier that two and half ovum are bonding, bonding half good ovum merges liquid equilibrium number second to putting into, and puts into electric integration slot alternating-current moment location again, and its contact surface can be vertical with direction of an electric field automatically, and then applies direct current and induce fusion.With merging half good ovum to merging activation with donorcells again, owing to be to have merged two half ovum, volume need not to apply alternating-electric field than the more convenient operation of conference, directly gets final product with the round end glass needle somatocyte that sets right.
Embodiment
Fusion method in a kind of animal somatic cell nuclear transplantation of the present invention is characterized in that, may further comprise the steps:
(1), it is right two and half ovum to be put into the bonding formation ovum-ovum of the operation liquid that contains the 0.5-5mg/ml phytohemagglutinin;
(2), bonding good ovum-ovum was merged the liquid balance 1-3 minute to putting into;
(3), the ovum-ovum after the balance is merged putting into electric integration slot electricity, and apply alternating-current field to ovum-ovum to carrying out the moment location;
(4), will merge in good ovum-ovum contains donorcells to immigration the operation liquid and the bonding organizer-ovum of donorcells; Phytohemagglutinin concentration is 0.5-5mg/ml's in the operation liquid;
(5), bonding good body-ovum moved into merged in the exciting liquid balance 1-3 minute, be moved into again and carry out electricity in the integration slot and merge activation simultaneously.
Merge described in the above-mentioned steps (2) in the liquid, the 4-HEPES, the concentration that contain N.F,USP MANNITOL, concentration that concentration is 0.2 ∽ 0.5mmol/L and be 0.5 ∽ 2mmol/L are the Z 150PH of 0.01g/L.
Merge in the exciting liquid described in the above-mentioned steps (5): containing concentration 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 Mg Cl
26H
2O, concentration are that 0.5 ∽ 2mmol/L 4-HEPES, concentration are the 0.01g/L Z 150PH.
Further improvement of the present invention is, step (1) is preceding puts into the operation liquid 3-8 second of containing phytohemagglutinin respectively with said two and half ovum earlier carrying out.
Claims (4)
1. the fusion method in the animal somatic cell nuclear transplantation is characterized in that, may further comprise the steps:
(1), two and half ovum being put into phytohemagglutinin concentration is that the bonding formation ovum-ovum of operation liquid of 0.5-5mg/ml is right;
(2), bonding good ovum-ovum was merged the liquid balance 1-3 minute to putting into;
(3), the ovum-ovum after the balance is merged putting into electric integration slot electricity, and apply alternating-current field to ovum-ovum to carrying out the moment location;
(4), will merge in good ovum-ovum contains donorcells to immigration the operation liquid and the bonding organizer-ovum of donorcells;
(5), bonding good body-ovum moved into merged in the exciting liquid balance 1-3 minute, be moved into again and carry out electricity in the integration slot and merge activation simultaneously.
2. the fusion method in the animal somatic cell nuclear transplantation according to claim 1 is characterized in that, step (1) is preceding earlier puts into the operation liquid 3-8 second of containing phytohemagglutinin respectively with said two and half ovum carrying out.
3. the fusion method in the animal somatic cell nuclear transplantation according to claim 1; It is characterized in that; Merge described in the step (2) in the liquid, the 4-HEPES and the concentration that contain N.F,USP MANNITOL, concentration that concentration is 0.2 ∽ 0.5mmol/L and be 0.5 ∽ 2mmol/L are the Z 150PH of 0.01g/L.
4. the fusion method in the animal somatic cell nuclear transplantation according to claim 1 is characterized in that, merge in the exciting liquid described in the step (5): containing concentration 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/LMg Cl
26H
2O, concentration are that 0.5 ∽ 2mmol/L 4-HEPES, concentration are the 0.01g/L Z 150PH.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012101944035A CN102703511A (en) | 2012-06-10 | 2012-06-10 | Fuse method for animal body nucleus transfer |
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| CN2012101944035A CN102703511A (en) | 2012-06-10 | 2012-06-10 | Fuse method for animal body nucleus 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 |
| CN112218534A (en) * | 2018-04-15 | 2021-01-12 | 以色列国家农业和农村发展农业研究组织沃尔坎尼中心 | Entomopathogenic nematode (EPN) species as biological delivery systems |
| CN113174410A (en) * | 2021-04-21 | 2021-07-27 | 广西壮族自治区畜牧研究所 | Method for improving the means of donor-acceptor cell adhesion in manual cloning procedures |
-
2012
- 2012-06-10 CN CN2012101944035A patent/CN102703511A/en active Pending
Cited By (5)
| 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 |
| 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 |
| CN112218534A (en) * | 2018-04-15 | 2021-01-12 | 以色列国家农业和农村发展农业研究组织沃尔坎尼中心 | Entomopathogenic nematode (EPN) species as biological delivery systems |
| US11992014B2 (en) | 2018-04-15 | 2024-05-28 | The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) | Entomopathogenic nematodes (EPN) species as a biological delivery system |
| CN113174410A (en) * | 2021-04-21 | 2021-07-27 | 广西壮族自治区畜牧研究所 | Method for improving the means of donor-acceptor cell adhesion in manual cloning procedures |
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Application publication date: 20121003 |