CN102344941A - Method for breeding cloned animal or transgenic cloned animal - Google Patents
Method for breeding cloned animal or transgenic cloned animal Download PDFInfo
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- CN102344941A CN102344941A CN2010102411560A CN201010241156A CN102344941A CN 102344941 A CN102344941 A CN 102344941A CN 2010102411560 A CN2010102411560 A CN 2010102411560A CN 201010241156 A CN201010241156 A CN 201010241156A CN 102344941 A CN102344941 A CN 102344941A
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Abstract
The invention discloses a method for breeding a cloned animal or a transgenic cloned animal. The invention further provides a method for breeding a cloned animal embryo or a transgenic cloned animal embryo. The method provided by the invention comprises the following steps of: (1) obtaining a denucleated oocyte; (2) fusing a donor cell with the denucleated oocyte to obtain a reconstructed embryo; (3) activating the reconstructed embryo in vitro; and (4) culturing the activated reconstructed embryo. The method is characterized in that: in the step (3), in-vitro activation comprises the following specific steps of: treating the reconstructed embryo twice with a calcium ion activator; and activating the treated reconstructed embryo in a protein synthesis inhibitor or a protein phosphorylation inhibitor to obtain the reconstructed embryo. As proved by an experiment, due to the adoption of the technical scheme, the seven-month pregnancy rate, the development rate and the survival rate after birth of cloned cattle are 44 percent, 33 percent and 100 percent respectively.
Description
Technical field
The present invention relates to a kind of method of cultivating cloned animal and transgenic animal.
Background technology
Mammalian somatic cell clone comprises steps such as the maturation cultivation, donorcells cultivation, nuclear transplantation operation, cytogamy, embryo culture of ovocyte.When conventional somatic cell clone, the acceptor ovocyte be take out from the ovary surface the ovarian cumulus ovocyte, after maturation in vitro is cultivated 20-22hr (ox, sheep etc.), 40-44hr (pig), remove cumulus cell.And then the ovum of selection discharge first polar body, after its nuclear substance removal, as recipient cytoplasm.Handle the ovocyte that obtains like this, its first polar body is easy to depart from the genetic material of ovum, and in the blind suction stoning process that with PB1 is sign, the stoning rate reduces.If use fluorescence dye and be aided with ultraviolet irradiation, be prone to cause injury to ovum, influence embryo quality.
Select the good somatocyte of form to inject the ovum week crack of enucleation oocyte, somatocyte is incorporated in the ovum, form reconstructed embryo through electric pulse stimulation.In the meantime, merge embryo, just move on in the embryo medium and cultivate without any processing.Then, reconstructed embryo makes its activation through chemical treatment again.Activiation method is a calcium ion activated dose of conjugated protein inhibitor treatment.Cultivate changing embryo medium over to through the embryo who activates processing.
The deficiency of conventional clone operations is that the easy occurrence positions drift of the karyomit(e) of reconstructed embryo, diffusion phenomena cause the improper diploid rate of embryo to increase, and form a plurality of micronucleus, influence fetal development and embryo quality.The final cloning efficiency that influences.
Summary of the invention
The object of the present invention is to provide a kind of method of cultivating cloned animal embryo or transgenic and cloned animal embryo.
Aforesaid method provided by the invention comprises the steps:
1) obtains stripped enucleation oocyte; 2) donorcells and the fusion of said enucleation oocyte of exsomatizing obtained reconstructed embryo; 3) said reconstructed embryo is carried out external activation; 4) reconstructed embryo that activates is cultivated; It is characterized in that: in the step 3); Said external activation comprises said reconstructed embryo with the calcium ion activated dose of processing of carrying out more than 2 times; The reconstructed embryo that to handle activates in protein synthesis inhibitor or protein phosphorylation inhibitor then; The reconstructed embryo that obtains activating is cultivated the reconstructed embryo of said activation and is obtained said cloned animal embryo or transgenic and cloned animal embryo.
In one embodiment, the number of times of above-mentioned processing is 2 times; Said processing comprises the steps:
A) said reconstructed embryo is placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10-15 minute;
B) on the basis of step a), said reconstructed embryo was placed in said calcium ion activated dose 5 minutes.
In another embodiment, the number of times of above-mentioned processing is 3 times; Said processing comprises the steps:
A) said reconstructed embryo is placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10-15 minute;
B) on the basis of step a), said reconstructed embryo was placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10 minutes;
C) on the basis of step b), said reconstructed embryo was placed in said calcium ion activated dose 2 minutes.
Said embryo medium is in CR1aa nutrient solution or SOFaa nutrient solution, to add the nutrient solution that BSA obtains; Said BSA is 0.4g/100ml at the final concentration of said embryo medium; Said culture condition is 38.5 ℃ and 5%CO
2The solvent of said CR1aa nutrient solution and SOFaa nutrient solution is water, and solute is respectively shown in table 1 and table 2.
The solute of table 1.CR1aa nutrient solution
| Solute | Concentration (/L) |
| NaCl | 6.7g |
| KCl | 0.23g |
| NaHCO3 | 2.2g |
| Gentamicin (Sigma, G1397) | 50μL |
| Lactic acid half calcium (hemicalcium lactate) | 0.55g |
| Sodium.alpha.-ketopropionate (pyruvate) | 0.04g |
| L-glutaminate (L-glutamine) | 0.146g |
| Non-essential amino acid (V/V) (Sigma, M7145) | 1000μL |
| Indispensable amino acid (Sigma, B6766) | 2000μL |
The solute of table 2.SOFaa nutrient solution
| Solute | Concentration (/L) |
| NaCl | 6.33g |
| KCl | 0.533g |
| CaCl2 | 0.19g |
| KH2PO4 | 0.162g |
| MgSO4.7H2O | 0.18g |
| NaHCO3 | 2.1g |
| Na?pyruvate | 0.036g |
| Na?lactate | 0.37g |
| Glutamine | 0.146g |
| Myo-inositol | 0.05g |
| Non-essential amino acid (V/V) (Sigma, NM7145) | 1000μL |
| Indispensable amino acid (Sigma, B6766) | 2000μL |
Said calcium ion activated dose is ionomycin, calcium ion carrier A 23187, ethanol or strontium chloride; Said ionomycin is present in the nutrient solution one; The final concentration of said ionomycin in said nutrient solution one be 2.0-5.0 μ mol/L preferably;
Said protein synthesis inhibitor is cycloheximide (cycloheximide); Said protein phosphorylation suppressor factor is 6-dimethylamino-purine (6-DMAP);
Said cycloheximide and said 6-dimethylamino-purine are present in respectively in nutrient solution two and the nutrient solution three, and said nutrient solution one, nutrient solution two, nutrient solution three are all identical with above-mentioned embryo medium; The final concentration of said cycloheximide in said nutrient solution two is to be preferably 10 μ g/mL; The final concentration of said 6-dimethylamino-purine in said nutrient solution three is to be preferably 1.9-2.1mmol/L; The said activated time is 4-6 hour.
Preferably; Aforesaid method also comprises step 2) and step 3) between the NSC 630176 treatment step; Said NSC 630176 is handled and may further comprise the steps: with step 2) in said reconstructed embryo move in the embryo medium that contains NSC 630176 and activate preceding cultivation; Incubation time is 1-6 hour before the said activation, preferably 2-3 hour;
The said embryo medium that contains NSC 630176 preferably adds the nutrient solution that BSA and NSC 630176 obtain in the CR1aa nutrient solution; The final concentration of BSA and NSC 630176 is for as follows 1. or 2. in the said embryo medium that contains NSC 630176:
1. BSA is that 0.3-0.5g/100ml, NSC 630176 are 1.0-10ng/ml;
2. BSA is that 0.4g/100ml, NSC 630176 are 10ng/ml;
Culture condition is 38.5 ℃ and 5%CO before the said activation
2The solvent of said CR1aa nutrient solution is a water, and solute is as shown in table 1.
Said NSC 630176 is Trichostatin A (TSA) preferably.
In step 1), said acquisition enucleation oocyte comprises following three steps:
I) when the ovarian cumulus-ovocyte that exsomatizes-complex body maturation in vitro is cultivated 12-18 hour, remove cumulus cell, obtain removing the ovocyte of cumulus cell;
II) with step I) ovocyte of the removal cumulus cell that obtains continues maturation in vitro and cultivates, and obtains having the non-nucleus egg mother cell of treating of first polar body;
III) with Step II) non-nucleus egg mother cell of treating that obtains carries out stoning, obtains enucleation oocyte.
Step II) in, the time that said continuation maturation in vitro is cultivated is 4-8 hour;
Step I) and Step II) in, it is in the M199 nutrient solution, to add in the ripe liquid that following material obtains to cultivate that said maturation in vitro is cultivated:
Foetal calf serum, Sodium.alpha.-ketopropionate, follicular stimulating hormone, metakentrin, estradiol, penicillin and Streptomycin sulphate;
Wherein, the final concentration of the material of interpolation in said ripe liquid is respectively foetal calf serum 10% (volume percent), Sodium.alpha.-ketopropionate 0.38mM/L, follicular stimulating hormone 0.01U/mL, metakentrin 0.01U/mL and estradiol 1 μ g/mL, penicillin 50 μ g/mL and Streptomycin sulphate 100 μ g/mL.
Said maturation in vitro culture condition is: 38.5 ℃, 5%CO
2With the saturated humidity condition;
Wherein, the M199 nutrient solution is available from Geibco or HyClone company.
The arbitrary above-mentioned application of method in cultivating cloned animal or transgenic and cloned animal also belongs within protection scope of the present invention.
Above-mentioned animal can be ox, sheep, pig or mouse.
Experiment proof: obtain to adopt two-step approach provided by the invention can obtain the stoning rate (table 3) more than 95% in the enucleation oocyte step; The integrity that has farthest kept the ovocyte kytoplasm.Reconstructed embryo before TSA handle to activate can make being kept perfectly property of karyomit(e), has guaranteed to move into the integrity (Fig. 1) of the genetic material of nuclear, and after TSA handles, fetal development quality be improved (the blastomere number brought up to 111.3 from 87.6 in 7 days) (table 4).Activate in the step; Reconstructed embryo is after the ionomycin activation is handled 2 times, and the clone embryos quality improves, behind clone embryo transplantation such as receptor cow (ewe); Pregnancy rate, grow the rate that expires, calf (lamb) survival rate significantly improves, the abnormal rate of new-born calve (lamb) significantly reduces.Somatic cell clone and transgenic and cloned animal production efficiency (table 5-table 10) have been significantly improved.
Description of drawings
Fig. 1 handles the influence to reconstructed embryo chromatin stability before activating for TSA.
Embodiment
Used raw material in the following example all can not obtain from commercial sources if there is special instruction.
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be ordinary method like no specified otherwise.
The somatic cell clone of embodiment 1, Japan and ox
One, somatic cell culture and processing
1, Japan and ox adult fibroblastic separate, cultivation
Get stripped Japan that grows up from Inner Mongol rising sun company and the have sharp ears tissue of ox.The ear local skin is scraped hair with knife blade earlier, uses the alcohol disinfecting of 70%-75% again, cuts the about 1cm of rapid clip have sharp ears with tissue
3The tissue of size soaks 60s immediately in the alcohol of 70%-75%, in the sterilization PBS that contains 250U/mL penicillin and 250 μ g/mL Streptomycin sulphates, clean 2-3 time, puts into same solution, takes back the laboratory for 4 ℃.In Bechtop, ear tissue with after the PBS rinsing 2 times, is put into penicillin bottle in the 60mm culture dish, do not add any solution, with eye scissors repeated shear tissue block to about 1mm
3Size.After respectively cleaning 3 times with PBS and the DMEM (DMEM+20%FBS) that contains 20%FBS, the picking tissue block is inoculated in 2-3 the 60mm culture dish, and every ware is inoculated 20-30 tissue block approximately, puts into CO
2Incubator, at 37 ℃, 5%CO
2Under the saturated humidity condition, dry up and cultivate 3hr, add then 5mL contain DMEM+20%FBS carry out former be commissioned to train foster.When cell growth reaches 90% when converging, the sucking-off nutrient solution, PBS cleans 3 times with sterilization, adds the Digestive system of 1mL 0.25% trypsinase+0.02%EDTA, and 37 ℃ of digestion 2-3min add 2mL DMEM+10%FBS immediately and stop digestion.Went down to posterity according to 1: 4 then.
2, the separation of fetal fibroblast and cultivation
Take the live body modus operandi, in the uterus, strip 40 to 60 days fetus of gestation, take back the laboratory at 4 ℃.The organs such as head, hoof and internal organ of fetus are removed.Operation handlebar fetus according to step 1 shreds, and cultivates.
3, cell cryopreservation, recovery and cultivation
Cell in step 1,2 grows to and surpasses 80% when converging; Clean 3 times with 4-5mL PBS; Use 0.25% trypsinase, under 37 ℃ of conditions, digestion 3min; Piping and druming makes it to suspend; Collecting cell then, the centrifugal 5min of 1500rpm/min, abandoning supernatant; The re-suspended cell counting puts into 1 * 10 in each freeze pipe
6Individual/the mL cell, and add DMEM nutrient solution, 20%FBS, 10%DMSO.Earlier frozen pipe is put under 4 ℃ of conditions balance half an hour ,-20 ℃ are spent the night, and-80 ℃ are dropped into liquid nitrogen freezing after depositing 24hr.
The recovery of cell: from liquid nitrogen, take out frozen pipe, drop into 37 ℃ of water-baths immediately, treat that ice cube just melts, disinfect frozen pipe outer wall in alcohol after, place super clean bench immediately.Cell suspension in the frozen pipe is moved in the 10mL centrifuge tube, clean frozen pipe twice with nutrient solution, scavenging solution moves in the centrifuge tube, and room temperature is centrifugal, and (1500rpm/min 5min), abandons supernatant.Add the DMEM nutrient solution re-suspended cell that contains 10%FBS and be inoculated in the 60mm Tissue Culture Dish, 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, when treating that cell grows to about 70-80% and converges, go down to posterity or be used for nuclear transplantation.The nuclear transplantation all cells is 3-12 generation.
Two, the processing of ovocyte and nuclear transplantation operation
1, the processing of ovocyte is cultivated as contrast by the maturation that novel method of the present invention is carried out, the while is carried out ovocyte with ordinary method:
1) as the ordinary method of contrast: the ovary when bamboo top or milk cow that will exsomatize (taking from the Hu Shi slaughterhouse) with 37 ℃ PBS liquid cleaning three times after; Use diameter to extract the ovarian follicle of diameter as 2-8mm as the syringe needle of 0.7mm; Reclaim the ovarian cumulus-ovocyte-complex body (COCs) of uniform form, compact structure; It (is added the ripe liquid that following material obtains: foetal calf serum, Sodium.alpha.-ketopropionate, follicular stimulating hormone, metakentrin, estradiol, penicillin and Streptomycin sulphate with ripe liquid in the M199 nutrient solution; Wherein, The final concentration of material in said ripe liquid that adds is respectively foetal calf serum 10% (volume percent), Sodium.alpha.-ketopropionate 0.38mM/L, follicular stimulating hormone (bFSH) 0.01U/mL, metakentrin (bLH) 0.01U/mL and estradiol 1 μ g/mL, penicillin 50 μ g/mL and Streptomycin sulphate 100 μ g/mL; Wherein the M199 nutrient solution is available from Geibco or HyClone company) wash twice; Then ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid by 40-50 piece/hole, place 38.5 ℃, 5%CO
2In the incubator ripe cultivate 18-20h after, sophisticated ovocyte is put into the pipe that the contains 0.1% Unidasa 2-3min that vibrates, cumulus cell and ovocyte are broken away from fully.Then, select complete form, tenuigenin evenly and the ovocyte of discharging first polar body be cytosol receptor, also promptly obtained to treat non-nucleus egg mother cell.
2) method provided by the invention:
A, the first step: basic identical with ordinary method.In the ovarian follicle of the ovary surface 2-8mm that exsomatizes, extract COCs.With COCs in above-mentioned ripe liquid, wash 3-4 all over after, be one group with 40-50 COCs and put into 4 orifice plates that contain 0.5mL pre-equilibration maturation liquid, at 38.5 ℃, 5%CO
2With cultivate under the saturated humidity condition.When COCs maturation in vitro 15-16hr, remove cumulus cell with 0.1% Unidasa liquid vibration COCs 2-3min.Under stereoscope, remove the paramophia or the dead ovocyte of degenerating.Other ovocyte gets into next step processing.
B, second step: the first step is cultivated the ovocyte of the no ovarian cumulus obtain, put in advance at the ripe liquid of incubator equilibrated again, 50-60 piece of ovocyte put in every hole.Continuation is at 38.5 ℃, 5%CO
2With cultivate 6hr under the saturated humidity condition, the ovocyte that obtain to have first polar body this moment is for treating non-nucleus egg mother cell.
2, nuclear transplantation operation
The ovocyte that has first polar body that step 1 is obtained moves in the operation liquid (on the basis of M199, having added 10%FBS and 5.0 μ g/mL cytochalasin Bs); Under 200 power microscopes, hold ovocyte with fixed tube; The position of adjustment ovum makes its first polar body be positioned at 12 o'clock to 1 o'clock position of clock.Using internal diameter again is that the syringe of 20 μ m passes zona pellucida from about position at 2, and the karyomit(e) in the ovocyte of first polar body and below thereof is absorbed in the lump.Then, the form of choosing step 1 is good, the somatocyte of tenuigenin homogeneous, the about 12 μ m of diameter is as donor cell, and the hole that stays during along stoning with syringe is embedded into cell between archiblast film and the zona pellucida.Accomplish the making of reconstructed embryo.
The stoning result is as shown in table 3, and the stoning rate of two-step approach provided by the invention is apparently higher than ordinary method.
The blind suction method stoning efficient that table 3. ovocyte single stage method and two-step approach are cultivated
| Handle | Operation ovum number | Stoning ovum number | Stoning rate (%) |
| Ordinary method | 78 | 58 | 74.3 a |
| Two-step approach provided by the invention | 81 | 77 | 95.1 b |
a，b：P＜0.01。
Conventional single stage method is when the 20-22hr that cultivates, to remove cumulus cell.In removing the cumulus cell process, make first polar body deviate from the position of its original discharge easily, that is to say that first polar body departs from the karyomit(e) (matter) of ovum.In the blind suction stoning process that with the first polar body is sign, can cause the stoning rate to reduce.Can be prone to cause injury according to the karyomit(e) (matter) of removing ovum though use fluorescence dye and be aided with ultraviolet irradiation, influence embryo quality ovum.And the first step of two-step approach is to remove cumulus cell at the 15-16hr that ovum is cultivated.Later stage or latter stage that the ovocyte overwhelming majority of this moment is in initial meiosis, (anaphase I or telophase I, AI/TI), first polar body was just prominent to ovum week crack with the form of a kytoplasm projection.In the second step culturing process, first polar body is discharged and also to be broken away from ovocyte, but rests on the position of its original discharge all the time, and promptly first polar body is in the position with the karyomit(e) next-door neighbour of ovum always.When stoning, as long as first polar body and a little contiguous kytoplasm thereof are removed, the genetic material of ovum all can be removed, significantly improve the stoning rate, and farthest keep the integrity of archiblast.Table 1 has promptly compared single stage method and two-step approach is cultivated the situation that influences to the stoning rate.
In brief: the advantage of this inventive point is the stoning rate more than 95%; The integrity that has farthest kept the ovocyte kytoplasm.
3, cytogamy
The reconstruct ovum to be merged that is obtained by two-step approach provided by the invention in the step 2 is transferred in the embryo culture drop (CR1aa nutrient solution), put into incubator and recover about 0.5hr.Then, the reconstruct ovum is placed fusion liquid (the 0.27M N.F,USP MANNITOL+0.1mM CaCl of preheating
22H
2O+0.1mM MgSO
47 H
2O+0.05%BSA) in, equilibrium at room temperature 5min changes interpole gap then over to and is 1mm and is added with in the integration slot that merges liquid.The direction of adjustment reconstruct ovum; Make the contact surface of ovocyte and donorcells parallel with the two poles of the earth; Impose 120V/mm 2 times; 25 μ s; The square wave electric pulse (fusion instrument is the ECM-2001 of BTX company) that the interval is 100 milliseconds; Put into embryo medium after every group of 15 completion, put and recover in the incubator to check the fusion situation behind the 30min.After fusion treatment finishes the 2nd hour and the 4th hour are to reconstructed embryo chromatin study on the stability.
4, TSA (Trichostatin A, Trichostatin A) handles:
Behind the embryo's continuation cultivation 0.5h that has merged in the step 3, be moved in the embryo medium (CR1aa+0.4g/100ml BSA) that contains 10ng/ml TSA at 38.5 ℃, 5%CO
2Cultivate 2h under the condition.After fusion the 2nd hour and the 4th hour are investigated reconstructed embryo chromatin stability.
Reconstructed embryo chromatin study on the stability result in step 3 and the step 4 such as Fig. 1 (F2h: merge back 2hr; F4h: merge back 4hr; The TSA-representative is not handled and the direct control group of in step 3, investigating through TSA in the step 4; The TSA+ representative experimental group that TSA handles in step 4.DNA represents chromatin, and Tubulin represents tubulin) shown in, at control group, activating again behind the 4hr after merging, drift takes place the karyomit(e) of nuclear disperses; And the being kept perfectly property of karyomit(e) of TSA treatment group, so after the TSA intervention, guaranteed to move into the integrity of the genetic material of nuclear.
5, merging embryo activates and embryo culture
The fusion embryo that step 4 is obtained activates according to following two kinds of experimental group:
A organizes (activating 1 time): merge embryo with containing 5 μ M ionomycin (Sigma; I0634) nutrient solution (CR1aa+0.4g/100ml BSA) is handled 5min once; Entering contains 10 μ g/ml cycloheximide (cycloheximide) (Sigma; C7698) in the nutrient solution (CR1aa+0.4g/100ml BSA), at 38.5 ℃, 5%CO
2With cultivate 5hr under the saturated humidity condition, accomplish to activate handle;
B organizes (activating 2 times): merge embryo with the above-mentioned nutrient solution processing 5min that contains 5 μ M ionomycins once after, the embryo is changed in the nutrient solution (CR1aa+0.4g/100ml BSA) at 38.5 ℃ over to 5%CO
2Cultivate 10-15min in the incubator, handle 5min through the above-mentioned nutrient solution that contains 5 μ M ionomycins again, change over to afterwards in the above-mentioned nutrient solution that contains 10 μ g/ml cycloheximide, at 38.5 ℃, 5%CO
2With cultivate 5hr under the saturated humidity condition, accomplish to activate handle.
A and b are through the activated reconstructed embryo afterwards, and it is inferior to give a baby a bath on the third day after its birth with nutrient solution, the embryo is put into the 30 μ L that covered by Yellow Protopet 2A cultivate droplet (a CR1aa+0.4g/100ml BSA), 38.5 ℃, cultivate 48h under the saturated humidity condition; Then, it is to cultivate 7 days (day of activation is designated as the 1st day) in CR1aa+4% (V/V) the FCS drop that the embryo is transferred to the nutrient solution that is covered with the individual layer cumulus cell, and therebetween, every 48hr changes liquid once.Respectively in statistics spilting of an egg rate of the 48hr after the cultivation and 168hr statistics blastaea rate (dripping with the embryo's entering CR1aa+0.4g/100mlBSA cultivation after activating is 0 hour).
In order to investigate the TSA treatment effect of step 4; Step 3 and step 4 are obtained merging embryo has carried out conventional activation and has handled (also being a group experiment in the step 5); (the TSA processing is the fusion embryo that step 4 obtains in the table for result such as table 4; Control group is the fusion embryo that step 3 obtains) shown in; After TSA handled, the fetal development quality was improved.
Table 4.TSA handles the influence of Niu Ronghe embryo to fetal development
In the same row, a, b:P＜0.05; A, c:P＜0.01.
In a word: TSA handles has following 3 each function:
1) intervention of TSA can guarantee to move into the integrity of the genetic material of nuclear, guarantees that promptly the karyomit(e) of nuclear is in a fixed zone, does not scatter and drift can not take place, and Fig. 1 promptly shows the TSA treatment effect.
2) intervention of TSA can prolong fusion and activate the timed interval between handling, and can extend to 3-4hr, can not influence chromosomal integrity and fetal development and quality thereof.
3) TSA can influence the state of acetylation of histone as NSC 630176, also possibly play regulating effect to the ovocyte kytoplasm, and the reprogrammed of the somatic cell nuclear that helps moving into helps improving fetal development quality (as shown in table 4).
In order to investigate the difference that a organizes and b organizes in the step 5, the fusion embryo that step 4 is obtained activates according to a, two experimental group of b again.The result is as shown in table 5, and fusion rate, spilting of an egg rate and the blastaea rate (bringing up to 45.2% by 33.1%) of promoting, the therefore quality that can promote to clone the beef cattle embryo are handled in twice activation.
The quality that promotes clone beef cattle embryo is handled in twice activation of table 5, ionomycin (Ionomycin)
* in the same row, a, b:P＜0.05.
The somatic cell clone of embodiment 2, milk cow
Present embodiment is divided into 4 experiments to carry out:
Experiment one, experiment one are following 2 points with the difference of embodiment 1, and all the other steps are identical:
1, the donor nuclei cell of Li Tiing is from the smooth milk cow of Hess of the auspicious auspicious animal husbandry in Inner Mongol limited liability company, and stripped ovary is from the bamboo top of working as in Hu Shi slaughterhouse;
2, the disengaging time point of cumulus cell and ovocyte is cultivated to begin to separate in 18-20 hour by maturation and has been become ripe cultivation and just began in 4 hours to separate.
Experiment two, experiment two and experiment once difference be that the disengaging time point is cultivated to begin to separate in 4 hours by maturation and become ripe cultivation and began to separate in 8 hours that all the other steps are identical.
Experiment three, experiment three and experiment once difference be that the disengaging time point is cultivated to begin to separate in 4 hours by maturation and become ripe cultivation and began to separate in 16 hours that all the other steps are identical.
Experiment four, experiment four are following 2 points with the difference of experiment one, and all the other steps are identical:
1, disengaging time point is cultivated to begin in 4 hours to separate by maturation and has been become ripe cultivation and just began in 22 hours to separate;
2, except that activating the activation of also carrying out following c group according to a among the embodiment 1 and two experimental group of b:
A organizes (activating 1 time): identical with embodiment 1;
B organizes (activating 2 times): identical with embodiment 1;
C organizes (activating 3 times): merge embryo with the above-mentioned nutrient solution processing 5min that contains 5 μ M ionomycins once after, the embryo is changed in the nutrient solution (CR1aa+0.4g/100ml BSA) at 38.5 ℃ over to 5%CO
2Cultivate 10-15min in the incubator, handle 5min (for the second time) through the above-mentioned nutrient solution that contains 5 μ M ionomycins again, the embryo is changed in the nutrient solution (CR1aa+0.4g/100ml BSA) at 38.5 ℃ over to 5%CO
2Cultivate 10min in the incubator, handle 2min (for the third time) through the above-mentioned nutrient solution that contains 5 μ M ionomycins again, afterwards the embryo is changed in the above-mentioned nutrient solution that contains 10 μ g/ml cycloheximide, at 38.5 ℃, 5%CO
2With cultivate 5hr under the saturated humidity condition, accomplish to activate handle.
The result is as shown in table 6, and ionomycin repeatedly activates has remarkable promoter action to spilting of an egg rate, blastaea rate, hatched blastocyst number and blastomere number.
Clone embryos developmental rate and the embryo quality that improves different training method ovocytes handled in repeatedly activating of table 6, ionomycin
In the same test group, different subscript symbols, significant difference is remarkable.a，b：P＜0.05。
The somatic cell clone of embodiment 3, Du Boyang
Present embodiment is divided in outer growth of clone's idiosome and the body grows two experiments.
One, clone's idiosome is grown experiment outward and is following 2 points with the difference of embodiment 1, and all the other steps are identical:
1, donor nuclei cell of Li Tiing and stripped ovary are all from Du Boyang (Siziwang Banner match promise animal husbandry scientific & technical corporation);
2, the CR1aa nutrient solution has all changed the SOFaa nutrient solution into.
SOFaa nutrient solution prescription is seen table 2.
The result is as shown in table 7, and spilting of an egg rate, blastocyst rate that ionomycin activates 1 time embryo all significantly are lower than 2 groups of ionomycin activation.Illustrate that 2 ionomycin handle the quality that has significantly improved the embryo.
Table 7, the ectogenesis of sheep somatic cell clone (SOFaa nutrient solution and cultivate altogether with cumulus cell)
* in the same row, a, b:P＜0.05.
Two, grow experiment in the body
The embryo that above-mentioned ectogenesis is obtained promptly moves in the acceptor ewe uterine tube of estrus synchronization behind vitro culture 20-30h (i.e. 2-to the 4-cell stage embryo of mark in the table 6), observes developmental state in its body.
The result is as shown in table 8, and developmental state shows in the body, and 40 days pregnancy rate, the lamb natality of 2 ionomycin activation treatment group are significantly higher than treatment group 1 time.
Table 8, somatic cell clone sheep testing data
When 2-to 4-cell stage, carry out embryo transfer.
4,2 ionomycin of embodiment activate growth in the body of handling the ox clone embryos, natality and survival rate
The difference of present embodiment and embodiment 1 is following:
1, the donor nuclei cell of Li Tiing is from the Japan and the ox of rising sun company, and stripped ovary is from the bamboo top of working as in Hu Shi slaughterhouse;
2, to clone embryos through b group (activating 2 times) activate handle after, cultivate in the receptor cow uterus of blastaea immigration estrus synchronization of 7 days (activation finish dealing with then begin to cultivate be 0 hour).The pregnancy rate of adding up 7 months, grow expire rate, survival rate.
The result is as shown in table 9,7 months pregnancy rate, grow the rate that expires, birth back survival rate is respectively 44%, 33% and 100%.
Table 9, beef cattle clone embryo transplantation efficient and calf survival rate
5,2 ionomycin of embodiment activate the influence of handling growing in the down producing goat clone embryos body
The difference of present embodiment and embodiment 3 is:
1, the donor nuclei cell of Li Tiing is from the Inner Mongolia White Cashmere Goat ram of the Inner Mongol one down producing goat sheep stud, and stripped ovary is from the local goat in Hu Shi slaughterhouse;
2, the goat ovocyte with maturation in vitro 20-22h is a cytosol receptor;
3, to merge embryo through ionomycin activate handle 2 times after, cultivate 20-30h at the SOFaa nutrient solution, embryo transfer in the acceptor ewe uterine tube of estrus synchronization.40 days pregnancy rate, grow the rate that expires, survival rate was respectively for 21.4%, 14.3% and 100% (like table 10).
Table 10, down producing goat clonogenic assay data
Therefore; Reconstructed embryo is after the ionomycin activation is handled 2 times, and the clone embryos quality improves, behind clone embryo transplantation such as receptor cow (ewe); Pregnancy rate, grow the rate that expires, calf (lamb) survival rate significantly improves, the abnormal rate of new-born calve (lamb) significantly reduces.Somatic cell clone and transgenic and cloned animal production efficiency have been significantly improved.
Claims (10)
1. a method of cultivating cloned animal embryo or transgenic and cloned animal embryo comprises the steps:
1) obtains stripped enucleation oocyte; 2) donorcells and the fusion of said enucleation oocyte of exsomatizing obtained reconstructed embryo; 3) said reconstructed embryo is carried out external activation; 4) reconstructed embryo that activates is cultivated; It is characterized in that: in the step 3); Said external activation comprises said reconstructed embryo with the calcium ion activated dose of processing of carrying out more than 2 times; The reconstructed embryo that to handle activates in protein synthesis inhibitor or protein phosphorylation inhibitor then; The reconstructed embryo that obtains activating is cultivated the reconstructed embryo of said activation and is obtained said cloned animal embryo or transgenic and cloned animal embryo.
2. the method for claim 1, it is characterized in that: the number of times of said processing is 2 times; Said processing comprises the steps:
A) said reconstructed embryo is placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10-15 minute;
B) on the basis of step a), said reconstructed embryo was placed in said calcium ion activated dose 5 minutes.
3. the method for claim 1, it is characterized in that: the number of times of said processing is 3 times; Said processing comprises the steps:
A) said reconstructed embryo is placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10-15 minute;
B) on the basis of step a), said reconstructed embryo was placed in said calcium ion activated dose 5 minutes; Again said reconstructed embryo is changed in the embryo medium and cultivated 10 minutes;
C) on the basis of step b), said reconstructed embryo was placed in said calcium ion activated dose 2 minutes.
4. like claim 2 or 3 described methods, it is characterized in that: said embryo medium is in CR1aa nutrient solution or SOFaa nutrient solution, to add the nutrient solution that BSA obtains; Said BSA is 0.4g/100ml at the final concentration of said embryo medium; Said culture condition is 38.5 ℃ and 5%CO
2The solvent of said CR1aa nutrient solution and SOFaa nutrient solution is water, and solute is respectively shown in table 1 and table 2.
5. like arbitrary described method among the claim 1-4, it is characterized in that: said calcium ion activated dose is ionomycin, calcium ion carrier A 23187, ethanol or strontium chloride; Said ionomycin is present in the nutrient solution one; The final concentration of said ionomycin in said nutrient solution one be 2.0-5.0 μ mol/L preferably;
Said protein synthesis inhibitor is a cycloheximide; Said protein phosphorylation suppressor factor is the 6-dimethylamino-purine;
Said cycloheximide and said 6-dimethylamino-purine are present in respectively in nutrient solution two and the nutrient solution three, and arbitrary described embryo medium is all identical among said nutrient solution one, nutrient solution two, nutrient solution three and the claim 2-4; The final concentration of said cycloheximide in said nutrient solution two is to be preferably 10 μ g/mL; The final concentration of said 6-dimethylamino-purine in said nutrient solution three is to be preferably 1.9-2.1mmol/L; The said activated time is 4-6 hour.
6. like arbitrary described method among the claim 1-5; It is characterized in that: said method also comprises step 2) and step 3) between the NSC 630176 treatment step; Said NSC 630176 is handled and may further comprise the steps: with step 2) in said reconstructed embryo move in the embryo medium that contains NSC 630176 and activate preceding cultivation; Incubation time is 1-6 hour before the said activation, preferably 2-3 hour;
The said embryo medium that contains NSC 630176 preferably adds the nutrient solution that BSA and NSC 630176 obtain in the CR1aa nutrient solution; The final concentration of BSA and NSC 630176 is for as follows 1. or 2. in the said embryo medium that contains NSC 630176:
1. BSA is that 0.3-0.5g/100ml, NSC 630176 are 1.0-10ng/ml;
2. BSA is that 0.4g/100ml, NSC 630176 are 10ng/ml;
Culture condition is 38.5 ℃ and 5%CO before the said activation
2The solvent of said CR1aa nutrient solution is a water, and solute is as shown in table 1;
Said NSC 630176 is Trichostatin A preferably.
7. like arbitrary described method among the claim 1-6, it is characterized in that: in the step 1), said acquisition enucleation oocyte comprises following three steps:
I) when the ovarian cumulus-ovocyte that exsomatizes-complex body maturation in vitro is cultivated 12-18 hour, remove cumulus cell, obtain removing the ovocyte of cumulus cell;
II) with step I) ovocyte of the removal cumulus cell that obtains continues maturation in vitro and cultivates, and obtains having the non-nucleus egg mother cell of treating of first polar body;
III) with Step II) non-nucleus egg mother cell of treating that obtains carries out stoning, obtains enucleation oocyte.
8. method as claimed in claim 7 is characterized in that: Step II), the time that said continuation maturation in vitro is cultivated is 4-8 hour;
Step I) and Step II) in, it is in the M199 nutrient solution, to add in the ripe liquid that following material obtains to cultivate that said maturation in vitro is cultivated:
Foetal calf serum, Sodium.alpha.-ketopropionate, follicular stimulating hormone, metakentrin, estradiol, penicillin and Streptomycin sulphate;
Wherein, the final concentration of the material of interpolation in said ripe liquid is respectively foetal calf serum 10% (volume percent), Sodium.alpha.-ketopropionate 0.38mM/L, follicular stimulating hormone 0.01U/mL, metakentrin 0.01U/mL and estradiol 1 μ g/mL, penicillin 50 μ g/mL and Streptomycin sulphate 100 μ g/mL;
Said maturation in vitro culture condition is: 38.5 ℃, 5%CO
2With the saturated humidity condition.
9. the application of arbitrary described method in cultivating cloned animal or transgenic and cloned animal among the claim 1-8.
10. like arbitrary described method among the claim 1-8 or the described application of claim 9, it is characterized in that: said animal is ox, sheep, pig or mouse.
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