CN1170510C - Somatic cell process of cloning mammal - Google Patents
Somatic cell process of cloning mammal Download PDFInfo
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- CN1170510C CN1170510C CNB99119974XA CN99119974A CN1170510C CN 1170510 C CN1170510 C CN 1170510C CN B99119974X A CNB99119974X A CN B99119974XA CN 99119974 A CN99119974 A CN 99119974A CN 1170510 C CN1170510 C CN 1170510C
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Abstract
The present invention provides a method for obtaining reconstruction ova for cloning mammals. The method comprises the steps: providing an enucleated cytoplasm-supplied ovum mother cell and a nucleus-supplied cell; directly injecting the nucleus-supplied cell in the enucleated cytoplasm-supplied ovum mother cell by a microinjection method; forming a reconstruction ovum; activating the reconstruction ovum; forming an activated reconstruction ovum. The present invention also provides a method for cloning mammals by using the reconstruction ovum to generate a body cell. The methods of the present invention omit the steps of integrating cells and removing the cytoplasm of body cells, and therefore, the methods of the present invention are simple and have high efficiency.
Description
Technical field
The present invention relates to method that mammal is cloned, more specifically, relate to the method for coming cloning mammal with somatic cell.The invention still further relates to the method for preparing the reconstruct ovum that is used for cloning of mammalian animal.
Background technology
Mammal nuclear is transplanted, and not only is a kind of very important instrument in embryo's biological study, and also is a kind of method preferably to " good " embryo's (domestic animal) expanding propagation.
In mammal, used the early stage embryo's blastomere or the cultured cell of embryonic origin (embryonic stem cell abbreviates " ES cell " as) nucleus is provided, obtained corresponding cloned animal: sheep (using the 8-16 cell) [Willadsen S.M., et al, Nature, 1986]; Rabbit (using the 8-cell) [Stice S.L., et al, Bio.Reprod., 1989]; Pig (using the 2-8 cell) [Prather R.S., et al. Biol.Reprod., 1989]; Cattle (using the 2-32 cell) [Prather R.S., et al. J.Anim.Sci., 1987 ,]; Goat (using the 8-cell) [Zhang Yong, " external herding-herbvore domestic animal fascicle ", 1993]; Mice (using the ES-cell) [Tsunda Y., et al. Journal of Reproduction and Fertility., 1993].
In recent years, use somatic cell that nucleus is provided, also in some mammals, successfully obtained cloned animal.1997, people such as Willmult used galactophore epithelial cell and carry out nuclear transplantation and produced sheep many sharp [Willmut I., et al., Nature.1997].Subsequently, obtained dissimilar somatic cell clone animals again: (1) fetal fibroblast nuclear transplantation sheep [Schnieke A.E., et al., Science, 1997], (2) fetal fibroblast nuclear transplantation cattle [Gibelli J.B., et al.IBID, 1998], (3) uterine tubal epithelium nuclear transplantation cattle etc.Above nuclear transplantation animal all is method preparations that application cell merges.
In addition, people such as Wakayama T in 1988 utilize the another kind of somatic cell of mice-be cumulus cell, should remove by somatic Cytoplasm earlier, then nucleus is injected directly in the Mus ovum cell cytoplasm of enucleation, produced nuclear transplantation mice [Wakayama T., et al., Nature, 1988].
Yet the involved step of the method for described generation somatic cell clone animal is still very various, thus this area press for step still less, the method for cloned animal that efficient is higher.
Summary of the invention
Purpose of the present invention just provides a kind of step still less, the mammiferous method of usefulness somatic cell clone that efficient is higher.In the method, cytoplasmic processing is not removed in somatic cell, but somatic cell is injected directly in the non-nucleus egg mother cell, and saved the cell fusion step.
Another object of the present invention provides the method that a kind of new acquisition is used to prepare the mammiferous reconstruct ovum of somatic cell clone.In the method, cytoplasmic processing is not removed in somatic cell, but somatic cell is injected directly in the non-nucleus egg mother cell, and saved the cell fusion step.
In a first aspect of the present invention, provide a kind of somatic cell clone mammiferous preparation method, this method may further comprise the steps:
The mammal that enucleation is provided is for matter oocyte and the mammalian somatic cell that is used for for nuclear;
Use microinjection, will be used for being injected directly into described enucleation for the matter oocyte, form the reconstruct ovum for the mammalian somatic cell of nuclear;
Described reconstruct ovum is activated processing, form activated reconstruct ovum;
Described activated reconstruct ovum transfer in the fallopian tube of foster mother animal, is perhaps cultivated described activated reconstruct ovum in external or body, form reconstructed embryo, and then reconstructed embryo is transplanted to the intrauterine of foster mother animal;
Raise described foster mother animal, produce the mammal of somatic cell clone.
In another aspect of this invention, the method that provides a kind of acquisition to be used to prepare the mammiferous reconstruct ovum of somatic cell clone, this method may further comprise the steps:
The mammal that enucleation is provided is for matter oocyte and the mammalian somatic cell that is used for for nuclear;
Use microinjection, will be used for being injected directly into described enucleation for the matter oocyte, form the reconstruct ovum for the mammalian somatic cell of nuclear;
Described reconstruct ovum is activated processing, form activated reconstruct ovum.
Method of the present invention compared with prior art, its inventive point is: saved cytoplasmic treatment step is removed in somatic cell, but somatic cell is injected directly in the non-nucleus egg mother cell, and saved the cell fusion step.Owing to saved these steps, therefore cause the method for cloning mammal or produce the method for reconstruct ovum simpler and easy, efficient is higher.
Description of drawings
In Figure of description,
Fig. 1 has shown the mammiferous method of generation somatic cell clone in the prior art.
Fig. 2 has shown the mammiferous method of generation somatic cell clone of the present invention.
Fig. 3 has shown in the methods of the invention by operation and has obtained mammal ovocyte towards ovum.
Fig. 4 has shown and has carried out micrurgic photo in the methods of the invention.
Fig. 5 A-D has shown that mammal (sheep) oocyte is carried out enucleation to be operated, and enucleation oocyte is arrived in the somatic cell microinjection.Wherein, Fig. 5 A is the oocyte before enucleation; Fig. 5 B is the oocyte in the enucleation process; Fig. 5 C is expelled to somatic cell before the enucleation oocyte (moving before the nuclear); Fig. 5 D is expelled to somatic cell after the enucleation oocyte (moving after the nuclear).
Fig. 6 has shown to be grown by somatic cell clone and has formed body early embryo.
Fig. 7 A and 7B have distinguished sheep mammar gland epithelial cell (amplifying 200 times) that has shown adherent growth and the galactophore epithelial cell (amplifying 200 times) that suspends.
Fig. 8 has shown the flow chart that produces the somatic cell clone sheep in the present invention's one example.
The specific embodiment
Now, shown among the figure to produce the mammiferous method of somatic cell clone in the prior art referring to Fig. 1.This method may further comprise the steps:
(1) obtains to provide cytoplasmic oocyte (abbreviating " for the matter oocyte " as).
Usually, obtain to obtain by following two kinds of methods for the matter oocyte.The one, take out the immature egg blast cell from folliculus ovarii, and at maturation in vitro; The one, supply cytoplasmic mammal after superovulation is induced, II phase oocyte in its fallopian tube, going out.These methods that obtain for the matter oocyte all are the ordinary skill in the art.
(2) obtain donor cell (nuclear cell promptly is provided)
Usually, obtain donor cell and can adopt soma's (as skin) of adults, not phase somatic cell (as granular cell) etc., after related organization is shredded and digesting, be dispersed into single somatic cell,, form cell line then at In vitro culture.Also can directly be digested to single somatic cell.
(3) enucleation of oocyte
Generally, the nuclear genetic material of oocyte is used kernel removing needle machinery at microscopically remove, thereby obtain non-nucleus egg mother cell.
(4) move nuclear
The nuclear process of moving of the prior art has dual mode usually.First kind is to contain in the crack of cytoplasmic somatic cell immigration ovum week, and the somatic cell and the oocyte that will inject ovum week crack then merge by electricity irritation in merging liquid, form moving of fusion type and examine ovum (reconstruct ovum).Second kind is earlier somatic Cytoplasm to be removed, and somatic cell nuclear (not containing or contain a small amount of kytoplasm) is directly injected in the kytoplasm of enucleation oocyte again, forms moving of injection and examines ovum (reconstruct ovum).
(5) activate
Reconstruct ovum to forming in the step (4) is activated by electrostimulation or chemical method, forms the reconstruct ovum that is activated.
(6) interior or In vitro culture, perhaps directly transplanting of body
In order to observe the developmental state of reconstructed embryo, for the reconstruct ovum that is activated that forms in the step (5), it can be moved into fallopian tube and carries out of short duration cultivation in vivo, perhaps utilize feeder cells (feeder cell) (for example uterine tubal epithelium cell) to carry out In vitro culture.Then, the embryo who grows is moved in the uterus of foster mother animal (receptor).
In addition, the reconstruct ovum that is activated for forming in the step (5) also can directly move into it in fallopian tube of foster mother animal (receptor), treats its gestation farrowing.
(7) raise pregnant foster mother animal
Foster mother animal to gestation is carefully raised, to produce the somatic cell clone animal.The animal that bears is verified by the dna fingerprint analysis.
Now, shown the mammiferous method of generation somatic cell clone of the present invention among the figure referring to Fig. 2.The inventive method may further comprise the steps
(1) obtains for the matter oocyte
This step can be identical with the step (1) of above-mentioned prior art.Preferably, be that superovulation by female animal obtains for the matter oocyte.The method for preparing confession matter oocyte of this area routine is all applicable to the present invention.
(2) obtain for the nucleome cell
This step can be identical with the step (2) of above-mentioned prior art.The method for preparing confession nucleome cell of this area routine is all applicable to the present invention.
(3) enucleation of oocyte
This step can be identical with the step (2) of above-mentioned prior art.The pitting method of this area routine is all applicable to the present invention.
(4) move nuclear
In this step, the nucleome cell that supplies in the step (2) is directly injected non-nucleus egg mother cell with microinjection, form the reconstruct ovum.In the methods of the invention promptly, needn't remove kytoplasm, also needn't earlier somatic cell be moved in the ovum week crack and carry out fusion treatment again somatic cell.
(5) activate
Reconstruct ovum to forming in the step (4) is activated by electrostimulation or chemical method, forms the reconstruct ovum that is activated.The activation processing method of the routine in this area all can be used for the present invention.Usually, available electricity irritation is handled in the activation of reconstruct ovum and/or the chemical stimulation method is carried out.Preferably, the activation of reconstruct ovum is handled be selected from down group in the present invention: (i) the reconstruct ovum is at 0.2-0.4M mannitol, the hyclone of 3-5mg/ml, 0.08-0.12mM MgSO
4Activation liquid in, carry out 1-3 electricity irritation, voltage is 600-800V/cm, each electricity irritation continues the 20-80 microsecond; (ii) the reconstruct ovum is at 0.2-0.4M mannitol, the hyclone of 3-5mg/ml, 0.08-0.12mM MgSO
4Activation liquid in, carry out 1-3 electricity irritation, voltage is 600-800V/cm, each electricity irritation continues 20-80 microsecond (μ s), and then the following chemical method that carries out activates: promptly activate 5 minutes in 5-20 μ M ionomycin, and 1-5 μ M 6-DMAP (6-dimethylaminopurine, i.e. 6-dimethylaminopurine) activation 1-6 hour.
(6) interior or In vitro culture, perhaps directly transplanting of body
This step can be identical with the step (6) of above-mentioned prior art.This area routine carry out in the body or In vitro culture to the reconstruct ovum, the perhaps method of directly transplanting is all applicable to the present invention.
(7) raise pregnant foster mother animal
This step can be identical with the step (7) of above-mentioned prior art.Method of the raising gestation foster mother animal of this area routine and the method for check somatic cell clone animal are all applicable to the present invention.
By above-mentioned more as can be seen, method of the present invention has compared with prior art been saved cytoplasmic treatment step has been removed in somatic cell, but somatic cell has been injected directly in the non-nucleus egg mother cell, and has saved the cell fusion step.Owing to saved these steps, therefore cause the method for cloning mammal or produce the method for reconstruct ovum simpler and easy, efficient is higher.
The inventive method is applicable to various mammals, and preferably this mammal is inhuman mammal.The mammiferous example that can be used for the inventive method comprises (but being not limited to): sheep, cattle, pig, horse, Canis familiaris L., cat, tiger, monkey, rabbit, mice and rat.In addition, the people also can be used as a kind of mammal that is applicable to the inventive method.
The somatic cell that is applicable to the inventive method comprises the various somatic cells of having broken up, for example from the cell of various tissues (as skin, mammary gland, fallopian tube, muscle etc.) and organ (as ear, ovary etc.).The somatic example that can be used for the inventive method comprises (but being not limited to): Skin Cell, mammary glandular cell, oviduct cell, ear cell, gonad cell, epithelial cell, fibroblast, endotheliocyte, cumulus cell, muscle cell, neurocyte, osteoblast.
The method of various acquisition oocytes and oocyte is carried out the method that enucleation handles all can be used for the present invention in this area.In an example of the present invention, the mammal of enucleation is such acquisition for the matter oocyte: II phase oocyte in obtaining to be in by the superovulation method, carry out enucleation then and handle the mammal of acquisition enucleation for the matter oocyte.
In the methods of the invention, before somatic cell is by direct injection, can carry out pretreatment, so that it fully is dispersed into individual cells to it.For example, handle under the following conditions: after the somatic cell usefulness trypsinization of cultivating of 0.1-0.5%, use 1-10mg/L protease E again, 36-39 ℃, 3-8%CO
2Middle digestion 1-20 minute is used for direct injection then.
In the methods of the invention, somatic cell and oocyte can be from same animals, also can be from allogenic animal or heterogenous animal.
In the present invention,, can directly be moved in the fallopian tube of foster mother animal (receptor), be treated its gestation farrowing for the reconstruct ovum that is activated.Perhaps it can be moved into fallopian tube and carries out of short duration cultivation in vivo, perhaps utilize feeder cells (feeder cell) (for example uterine tubal epithelium cell) to carry out In vitro culture, form reconstructed embryo, so that observe the developmental state of reconstructed embryo.Reclaim this reconstructed embryo then,, treat its gestation farrowing its intrauterine of being transplanted to the foster mother animal.
Whether in the methods of the invention, also comprise verification step: promptly the animal to reconstructed embryo or birth carries out the dna fingerprint analysis, be cloned animal with checking.Also can verify whether be cloned animal according to the phenotype (for example Mao color and speckle) of cloned animal.
Describe the present invention in further detail below in conjunction with embodiment, should be understood that these embodiment only are used for purposes of illustration and are not used in the restriction scope of the invention.
Embodiment
A, method
Supply matter female animal superovulation technology (oocyte is provided):
Superovulation is meant adopts the preparation induced animal once to discharge the method for normal number of eggs ovulated several times.It is spent also synchronous much more than receptor to carry out once super row.[pregnant mare serum is urged gonadal hormone (PMSG) by hormone; Follicle stimulating hormone (FSH), follicle stimulating hormone releasing factor (LRH)] handle, make female animal be non-seasonal ovulation, and in once ovulating, can discharge more ovum.Animal that HORMONE TREATMENT is crossed, used still can be repeated super row again and be used 3-4 time, is different at aspects such as interval, hormone dosages, has utilized experimental animal so fully.There is multiple superovulation technology available.
The concrete grammar of Cai Yonging is in the present invention: every animal intramuscular injection Cloprostenol (PG, Shanghai family planning institute) 0.06-0.1mg/ time, inject after 10-14 days for the second time at interval, after injecting PG 10-14 days for the second time, begin super row, i.e. intramuscular injection FSH (Ningbo hormone products factory) at first, consumption divides 6 times by 7-12IU/kg (the allogenic animal consumption is not quite similar), 2 times/day, each 8-12 hour at interval.Injection LRH (Shanghai east wind pharmaceutical factory) when oestrusing in 24 hours at interval, 25ug/ time, 28-30 hour recovery oocyte behind the injection LRH.
For synchronous with the confession matter ewe that the oocyte kytoplasm is provided, acceptor ewe is 10-14 days intramuscular injection PG at twice at interval, and for preceding 24 hours of the super row's injection of matter ewe PG, receptor is also injected PG simultaneously, and time and the dosage of receptor injection LRH are identical with donor.
In mammalian somatic cell clone process, the In vitro culture of animal somatic cell and the foundation of cell line also are very important.According to kind and the requirement of required cloned animal, select tissue or organ to set up cell line.Its method is: tissue, organ or last noble cells become individual cells with trypsinization, cultivates, go down to posterity with conventional culture fluid such as M199 or RM1640 (adding 10% hyclone), and genetic analysis, freezing preservation or work are used for nuclear.
For matter oocyte donor female animal behind injection LRH 26-30 hour, carry out the collection of oocyte.Collection is the method by operation, abdominal part female animal is made a kerf, the fallopian tube of female animal and ovary are exposed to external, use that (F10 culture fluid+5%BSA) go out the oocyte (see figure 3) from fallopian tube is cultivated the oocyte of collecting in the CZB culture fluid towards ovum liquid.
The oocyte enucleation
Oocyte is moved in the operation liquid, and at microscopically, with holding fixedly oocyte of ovum pin, the polar body that oocyte is discharged is in 3 positions of clock and watch, and kernel removing needle faces or departs from polar body slightly and remove polar body and a part of kytoplasm (seeing Fig. 5 A and 5B).
The somatic cell of cultivating moves in the operation liquid after 0.25% trypsinization.Use protease E (1-10mg/L) again, 37 ℃, 5%CO
2Middle digestion 1-10 minute.After the washing, move in the operation liquid, again injection cell is advanced (Fig. 5 C and 5D) in the non-nucleus egg mother cell, form the reconstruct ovum.
The activation of reconstruct ovum
The reconstruct ovum adopts two kinds of methods to activate: the one, the reconstruct ovum is being activated liquid (0.3M mannitol, 4mg/ml hyclone, 0.1mM MgSO
4) in carry out electricity irritation (voltage 600-800V/cm, at every turn continue 40 microseconds (μ s)) after, the reuse chemical method activates (10 μ M ionomycins, 5 minutes+2 μ M 6-DMAP 2-6 hour).The reconstruct ovum moves in the normal CZB culture fluid and cultivates after activating.Reaction temperature is generally in 37 ℃ ± 2 ℃ scopes.
Use the method for operation, activated reconstruct ovum is sucked ovum shifting tube, ovum shifting tube is inserted from the fallopian tube horn mouth, ovum is moved in the fallopian tube, connect the portion's of drawing suture ligation at fallopian tube and uterus subsequently.After cultivating 6 days in vivo, cut fallopian tube, go out embryo's (being reconstructed embryo), observe the fetal development situation with culture fluid.
Embryo transfer
The reconstructed embryo of growing is moved into and synchronous foster mother animal (receptor) intrauterine of fetal development.Ultrasound diagnosis gestation whether is raised meticulously after 1 month, treats its farrowing.
The DNA of animal, the animal that donor cell is provided and receptor sheep of birth is carried out fingerprint analysis, whether verify cloned animal.
B, material
Used in the present invention M199 culture medium, RM1640 culture medium, F10 culture medium and CZB culture medium etc. all are culture medium (culture fluid) conventional in this area.Wherein, the prescription of CZB culture fluid is as follows:
Table 1, CZB culture fluid
| Composition | Molecular weight | mM | Grams per liter | Gram/100ml |
| NaCl | 58.44 | 81.62 | 4.7698 | 0.47698 |
| KCl | 74.55 | 4.83 | 0.3601 | 0.03601 |
| KH 2PO 4 | 136.09 | 1.18 | 0.1606 | 0.01606 |
| MgSO 4 | 120.4 | 1.18 | 0.1421 | 0.01421 |
| NaHCO 3 | 84.01 | 25.12 | 2.1103 | 0.21103 |
| CaCl 2·2H 2O | 147.0 | 1.70 | 0.2499 | 0.02499 |
| Sodium lactate | 112.1 | 31.30 | 3.5087 | 0.35087 |
| Sodium Pyruvate | 110.0 | 0.27 | 0.0297 | 0.00297 |
| EDTA (disodium salt) | 372.24 | 0.11 | 0.0409 | 0.00409 |
| Glutamine | 146.1 | 1.00 | 0.1461 | 0.01461 |
| BSA(mg/ml) | 5.00 | 5.0000 | 0.50000 | |
| Benzylpenicillin (IU/ml) | 1580IU/mg | 100.0 | 0.0630 | 0.00630 |
| Streptomycin (mg/ml) | 0.70 | 0.7000 | 0.07000 |
Embodiment 1
Use from the galactophore epithelial cell of Saanen goat and clone Saanen goat
In this embodiment, used concrete technology path as shown in Figure 8.Particularly, may further comprise the steps:
1, galactophore epithelial cell obtains and cultivates
Use operation method, sterilely get about 1cm
2Mammary gland tissue, in super-clean bench with PBS wash, shred, 0.125% trypsinization 0.5-3 hour, it is centrifugal that sterile gauze filters the back, precipitation is cultivated in four orifice plates with M199 culture fluid (containing 10%FBS) suspension.After treating that cell covers with, go down to posterity, freezing or be used for for nuclear.
2, super row of goat and receptor sheep is synchronous:
The super row of goat: every animal intramuscular injection PG, 0.06-0.1mg/ it is inferior, inject after 10-14 days for the second time at interval, after injecting PG 10-14 days for the second time, begin super row, i.e. intramuscular injection FSH at first, consumption divides 6 times by 7-12IU/Kg (the allogenic animal consumption is not quite similar), 2 times/day, each 8-12 hour at interval.24 hours at interval, injection LRH when oestrusing 25ug/ time, did suitably to adjust according to the body weight of sheep.
The receptor sheep synchronously: for synchronous with the confession matter ewe that the oocyte kytoplasm is provided, acceptor ewe is 10-14 days intramuscular injection PG at twice at interval, injecting PG preceding 24 hours for the super row of matter ewe, receptor is also injected PG simultaneously, time and the same donor of dosage of receptor injection LRH.
3, the recovery of oocyte:
After 28-30 hour, reclaim oocyte (Fig. 3) behind the goat injection LRH by operation.By the conventional cropping of operation, sterilize, open the abdominal cavity, pull out uterus and fallopian tube, with No. 7 syringe needles of 10ml syringe, be F10 culture fluid+5%BSA towards ovum liquid.Being rushed out intratubal ovum is connected in the cup of garden.Under stereoscopic anatomical lens, the oocyte that detects is placed the CZB culture fluid, at 37 ℃, cultivate in 5% CO2 gas incubator.
4, the enucleation for the matter oocyte supplies the nucleome cell with injection
Oocyte was moved in the CZB culture fluid (table one) that contains the 7.5ug/ml cytochalasin B 20 minutes, move to again in the CZB culture fluid that contains 20mM HEPES.To be digested to spheric galactophore epithelial cell simultaneously also moves in this liquid.With oocyte enucleation (Fig. 5 A and 5B).Galactophore epithelial cell is directly injected in the kytoplasm of oocyte after the enucleation, the cell of formation is referred to as reconstruct ovum (Fig. 5 C and 5D) again.The reconstruct ovum places CZB liquid to cultivate 30 minutes, activates processing then.
5, activation and embedding:
The reconstruct ovum is placed the CZB culture fluid that contains cytochalasin B (7.5ug/ml)+ionomycin (10 μ M), cultivated 5 minutes for 37 ℃.Then, move among the CZB that contains cytochalasin B (7.5ug/ml)+6-DMAP (2 μ M), in 37 ℃, sessile drop method was cultivated 2-6 hour.Reconstruct ovum after the activation moves in the CZB culture fluid, carries out embedding.
Double-layer embedment is adopted in embedding.Used embedding liquid is 0.8% and 1.0% agarose (low melting point) solution.This embedding liquid is like this preparation: in 10ml cone-shaped glass centrifuge tube an amount of agarose mixed with normal saline and boil, dissolve extremely fully and be placed on 41 ℃, water-bath is stand-by.
During embedding, in a plastics plate, add and contain Ca
2+, Mg
2+PBS liquid, add hyclone in PBS liquid bottom with suction pipe, the oocyte for the treatment of embedding is moved into the serum layer, it is sunk naturally, diapire to be positioned at, sucking-off.Move into then in another little plate that contains 0.8% agarose of just having poured out, the position of oocyte is moved (playing cleaning function) 2-4 time, evenly inhale ovum (certain distance is arranged between ovum and the ovum, but mutually by not being too far away) again, move among the PBS.Allow its natural coagulation, cut apart, change the big slightly suction pipe of a bore, carry out second layer embedding by same procedure, difference is: the agarose concentration of embedding liquid is 1.0%.
6, the culturing in vivo of reconstruct ovum and recovery
Use the method for operation, the reconstruct ovum after the embedding is sucked ovum shifting tube, ovum shifting tube is inserted from the fallopian tube horn mouth, in reconstruct ovum shift-in fallopian tube, subsequently in fallopian tube and the suture ligation of uterus connecting portion.After cultivating 6 days in vivo, cut fallopian tube, go out embryo's (reconstructed embryo), observe the fetal development situation with culture fluid.
7, the transplanting of reconstructed embryo
The reconstructed embryo of growing is moved into and the synchronous receptor intrauterine of fetal development.Ultrasound diagnosis gestation whether is raised meticulously after 1 month, treats its farrowing.
8, cloned animal checking
With the animal of birth, provide for the animal of nucleome cell and the DNA of foster mother sheep and carry out fingerprint analysis, whether verify cloned animal.
The result is as follows:
(1) superovulation:
| Super row goat sum | The development of ovary (diameter) | Ovulation is counted | Reclaim the ovum number | |||
| ＞2cm | ＞1.5cm | ＞1cm | Do not grow | |||
| 10 | 4 | 2 | 3 | 1 | 102 | 86 |
Super row's effect:
Development of ovary rate is (in ovary diameter＞1cm): 90% (9/10)
Average every goat number of eggs ovulated: 10.2 pieces (102 pieces/10)
Average every goat is reclaimed the ovum number: 8.6 (86/10)
Reclaim ovum efficient: 84% (86/102)
By above result as can be seen, the goat development of ovary is good, reclaims ovum efficient height, but average every goat number of eggs ovulated is low slightly.
(2) receptor synchronization of estrus: after the PG processing, the sync rates of sheep is 78% (12/16).
(3) Somatic Cell Culture: set up Saanen goat galactophore epithelial cell system, uterine tubal epithelium cell and granular cell system.Carried out the Chromosome number analysis in generation in the process.Observed 100 division phases, 2 times of chromosomes account for more than 70%,
The used galactophore epithelial cell of this test is primary cell (seeing Fig. 7 A and 7B), does not also carry out freezing and hungry the processing.
(4) enucleation and somatic cell injection:
The total ovum number of enucleation: 75 pieces, wherein survive 73 pieces, survival rate: 97% (73/75)
Survive the ovum number after the somatic cell injection: 68 pieces
Reconstruct survival rate of ovum: 93% (68/73)
(5) activate and the interior of short duration cultivation of body:
The chemokinesis method is adopted in the activation of reconstruct ovum, and the embryo after the activation moves into and cultivates in the common goat fallopian tube after 6 days, and operation is reclaimed, and observes the reconstructed embryo developmental state.
(6) embryonic development and transplanting
1, reconstructed embryo early development information slip
| Transplant the ovum number | Reclaim the ovum number | Reconstruct egg development rate (%) | |||
| The 2-4 cell | The 32-64 cell | Fructus Mori | In early days+late period blastaea | ||
| 68 | 46 | 6 | 12 | 5 | 10 |
2, transplant: will grow to the embryo transfer more than 32 cells to the receptor intrauterine.Every receptor moves into 4 pieces.
(7) to embryo's checking
To above-mentioned Fructus Mori stage and early stage/late period blastaea stage embryo that is in, the cell that takes a morsel carries out pcr amplification (dna fingerprint analysis), and the result shows the identical of fingerprint and Saanen goat.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (4)
1. an acquisition is used to prepare the method for the reconstruct ovum of somatic cell clone non-human mammal, it is characterized in that this method may further comprise the steps:
The non-human mammal that enucleation is provided is for matter oocyte and the non-human mammal somatic cell that is used for for nuclear, and described oocyte and somatic cell are of the same race;
Use microinjection, will be used for being injected directly into described enucleation for the matter oocyte, form the reconstruct ovum for the non-human mammal somatic cell of nuclear;
Described reconstruct ovum is activated processing, form activated reconstruct ovum.
2. the method for claim 1, it is characterized in that described non-human mammal somatic cell is selected from down the cell of group: Skin Cell, mammary glandular cell, oviduct cell, ear cell, gonad cell, epithelial cell, fibroblast, endotheliocyte, cumulus cell, muscle cell, neurocyte, osteoblast;
Described somatic cell was handled before direct injection under the following conditions: after the somatic cell usefulness trypsinization of cultivating of 0.1-0.5%, use 1-10mg/L protease E again, and 36-39 ℃, 3-8%CO
2Middle digestion 1-20 minute is used for direct injection then;
And the activation of reconstruct ovum handled being selected from down group: (i) the reconstruct ovum is at 0.2-0.4M mannitol, the hyclone of 3-5mg/ml, 0.08-0.12mM MgSO
4Activation liquid in, carry out 1-3 electricity irritation, voltage is 600-800V/cm, each electricity irritation continues the 20-80 microsecond; (ii) the reconstruct ovum is at 0.2-0.4M mannitol, the hyclone of 3-5mg/ml, 0.08-0.12mM MgSO
4Activation liquid in, carry out 1-3 electricity irritation, voltage is 600-800V/cm, each electricity irritation continues the 20-80 microsecond, and then the following chemical method that carries out activates: promptly activate 5 minutes in 5-20 μ M ionomycin, and activate 1-6 hour at 1-5 μ M 6-DMAP.
3. the method for claim 1 is characterized in that, this non-human mammal is selected from: sheep, cattle, pig, horse, Canis familiaris L., cat, tiger, monkey, rabbit, mice and rat.
4. the purposes of the reconstruct ovum of the described method preparation of claim 1 is characterized in that, is used to prepare the somatic cell clone non-human mammal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB99119974XA CN1170510C (en) | 1999-11-08 | 1999-11-08 | Somatic cell process of cloning mammal |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB99119974XA CN1170510C (en) | 1999-11-08 | 1999-11-08 | Somatic cell process of cloning mammal |
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| Publication Number | Publication Date |
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| CN1294902A CN1294902A (en) | 2001-05-16 |
| CN1170510C true CN1170510C (en) | 2004-10-13 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB99119974XA Expired - Fee Related CN1170510C (en) | 1999-11-08 | 1999-11-08 | Somatic cell process of cloning mammal |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102318581B (en) * | 2011-08-31 | 2013-04-17 | 内蒙古农业大学 | Method for increasing sheep cloning efficiency |
| CN106755112B (en) * | 2016-12-27 | 2019-07-12 | 云南农业大学 | A method of Local Excellent domestic animal kind is cultivated using somatic cell clone technique |
| CN107460208B (en) * | 2017-09-08 | 2019-11-08 | 天津博裕力牧科技有限公司 | Mammalian Somatic Cloning method and the culture solution used |
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