CN1715401A - Method for breeding herd bull - Google Patents
Method for breeding herd bull Download PDFInfo
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- CN1715401A CN1715401A CN 200410049790 CN200410049790A CN1715401A CN 1715401 A CN1715401 A CN 1715401A CN 200410049790 CN200410049790 CN 200410049790 CN 200410049790 A CN200410049790 A CN 200410049790A CN 1715401 A CN1715401 A CN 1715401A
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- cytochalasin
- cycloheximide
- clone
- breeding
- bull
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The method of breeding herd bull includes the following steps: 1. establishing nucleus supplying somatocyte line with somatocyte of herd bull and performing synchronous treatment; 2. fusing the nucleus supplying somatocyte into denucleated egg mother cell to obtain recombinant embryo; 3. in vitro activating and cutlurig the recombinant embryo for 2-7 days to obtain mesembryo, transferring the mesembryo into cow womb in mating season to obtain somatocyte cloning herd bull. The present invention is one asexual propagation process with genetic matter from the same unity and the excellent genetic characteristic of the donor bull inherited, and has no need of descendant determination and short culture period and low cost.
Description
Technical field
The present invention relates to a kind of method of breeding breeding oxen in biological technical field and the improvement of breed field.
Background technology
Since first somatic cell clone animal " many jasmines " was born in the world, the somatic cell clone technique of domestic animal had obtained development rapidly as a new biotechnology, became the popular domain of world today's biotechnology research.People utilize different clone successively to obtain clened cows with different cloning process, clone sheep, and clone pig, the clone mouse, the somatic cell clone individuality of multiple domestic animal such as clone cat etc. and animal, and wherein the technology with the somatic cell clone ox is the most perfect.It is reported that the whole world has tens countries successively to cultivate the somatic cell clone domestic animal at present.Somatic cell clone technique establishing great theory significance and great application prospect.In theory, it has proved that the somatocyte that height has broken up still can recover its totipotency under certain condition.On using, utilize somatic cell clone technique can expand the good domestic animal of numerous proterties in a large number, not only can keep the good character of domestic animal, can select the sex of domestic animal again; Utilize somatic cell clone technique can cultivate transgenic animal, thereby improve the production efficiency of transgenic animal greatly, reduce production costs, shorten the cultivation time; Utilize somatic cell clone technique can effectively save animals on the brink of extinction; Somatic cell clone technique also provides unique research platform for fundamental researches such as molecular biology, developmental biology, regeneration biological; In addition, this technology has also been opened up a brand-new field for therapeutic cloning in the medical research.
Somatocyte that can be by adopting good high yield cow by vegetative means, expands the offspring of numerous high yield cow as donor fast, makes the genetic resources of the classic high yield cow in the world obtain the utilization of full blast.Clone technology forward industrialization commercialization direction develops, and livestock industry developed countries such as the U.S., Canada, Australia have all set up relevant biotech company, promotes cattle breeding of this country and the job development of expanding the group.
Relative other similar technology, the advantage that the somatic cell nuclear transfer technique big area expands numerous good domestic animal is: the 1) sex of animal that may command is produced increases substantially production efficiency; 2) the animal that produces in full accord with parent on proterties, can continue good character to greatest extent, this is all to be beyond one's reach by other any hereditary means; 3) can utilize ox to do the replace-conceive cow and produce milk cow, save production cost in a large number.Directly high yield cow being expanded the group fast by clone technology breeds.Production level of milk cow and milk quality are stepped to a new level.And cultivate a high-quality breeding oxen under the present condition is a very long and complicated process, at first from a large amount of offsprings that syngenesis produced, screen a collection of bull, analyze by the various production traitss again its offspring, just can obtain a high-quality breeding oxen, this approximately needs the time in about 5 years, and the best phase of breeding of a breeding oxen has only 7 years, so bull has only breeding the phase about 3 years after obtaining the progeny testing result, causes the value of high-quality breeding oxen high.
The innovation and creation content
The purpose of this invention is to provide a kind of method of breeding breeding oxen.
Method of breeding breeding oxen provided by the present invention may further comprise the steps:
1) sets up for nucleome clone with the somatocyte of breeding oxen, and carry out the same period and handle;
2) with merging through moving in the non-nucleus egg mother cell of the processing same period in the step 1), obtain reconstituted embryo for the nucleome cell;
3) with step 2) in the reconstituted embryo that obtains in external activation and cultivated 2-7 days, obtain blastaea, described blastaea is moved in the recipient cattle horn of uterus of estrus synchronization, obtain the somatic cell clone breeding oxen.
In the step 1), the described nucleome clone that supplies is preferably skin fibroblast line.Be treated to the described same period described nucleome clone serum starvation 2-4 days that supply.
Step 2) in, describedly being fused to electricity irritation and merging, can be 2.0-2.5kV/cm in field intensity specifically, and the burst length is 10-40 μ s, and pulse number is 1-3 time, and the recurrent interval is to carry out in the DC pulse field of 1s.
In the step 3), described external activation can adopt following two kinds of methods to carry out:
1) described external activation adopts B cytochalasin B and cycloheximide to handle, described treatment process is transferred to then and continues in the nutrient solution that contains the 10ug/ml cycloheximide to handle 4 hours for described reconstituted embryo was handled 1 hour in the nutrient solution that contains 0.25ug/ml B cytochalasin B and 10ug/ml cycloheximide.
2) described external activation adopts electricity irritation and B cytochalasin B and cycloheximide to unite activation method, described associating activated method is for earlier handling 10-40 μ s with described reconstituted embryo in field intensity is the DC pulse field of 2.0kV/cm, and then described reconstituted embryo handled 1 hour in the nutrient solution that contains 2.5ug/ml B cytochalasin B and 10ug/ml cycloheximide, be transferred to then and continue in the nutrient solution that contains the 10ug/ml cycloheximide to handle 4 hours.
Described B cytochalasin B is cytochalasin B or B cytochalasin B D.
The present invention breeds the method for breeding oxen, take somatocyte from the breeding oxen health of good quality and high output, in vitro culture and set up clone, after handling the same period, utilize somatic cell nuclear transfer technique (clone technology), somatocyte has been transplanted in the ovocyte of nuclear, and merge at the external use physical method with this ovocyte, reconstituted embryo after the fusion, external handle by electricity irritation and chemical substance cytochalasin (CD) and cycloheximide (CHX) after, reconstituted embryo is activated and in vitro culture external, wait to clone embryonic development and move in the receptor cow horn of uterus of estrus synchronization, transplant and carried out pregnancy check in back 90 days to blastula stage, treat that fetation is finished after, produce operation by spontaneous labor or real abdomen, thereby obtain and the on all four somatic cell clone breeding oxen of cell donor inherited character.The present invention is owing to adopt skin flbroblast as supplying the nucleome cell, therefore make the process operation of somatic cell clone convenient and simple, can not produce damage to donor, adopt electricity irritation and chemical substance (B cytochalasin B and cycloheximide) to activate and handle reconstituted embryo, the probability that makes reconstituted embryo develop into blastaea obtains bigger raising.
Method of breeding breeding oxen of the present invention is that vegetative propagation, its genetic material come from same individuality fully owing to reproductive process, so it has been inherited by the good inherited character of clone's donor bull, do not need to carry out progeny testing, shortened the cultivation time greatly, provide cost savings, will bring huge economic benefit.
Embodiment
Embodiment 1, breed breeding oxen
1. supply the foundation of nucleome clone
Get the ear skin tissue of holstein cow breeding oxen, clean behind ear's lower edge dorsal part unhairing with 70% alcohol wash, pick from ear's lower edge dorsal part with blade again that to get area be 1cm
2About skin, place 0 ℃ DMEM/F12 to transport the laboratory as early as possible back, with PBS and 70% alcohol wash number all over after shred into 1mm
3About fritter, DMEM/F12 plants piece in the 25cm that contains 1mL DMEM/F12+10%FBS after cleaning 2 times in batches
2Culturing bottle in, treat to add DMEM/F12+10%FBS to 6mL again after tissue block adherent is firmly, in 37 ℃, 5%CO
2Incubator is cultivated 6-7d, and every 2d changes liquid 1 time, treat that the cell growth converges after, with 0.25% trypsin trypsin) had digestive transfer culture 2-3 time, frozen with DMEM/F12+20%FBS+10%DMSO in batches.Like this, through former foster, the cultivation of going down to posterity of being commissioned to train, vitro culture operation such as freezing, set up breeding oxen for nucleome somatocyte system.
2. the maturation of ovocyte is cultivated
Collect the ovary of the ox that grows up from the slaughterhouse, place 30 ℃ physiological saline in 4h, to deliver to the laboratory, after cleaning three times in 37 ℃ the PBS liquid, is the ovarian follicle of 2-8mm with the diameter for the 0.7mm syringe needle extracts diameter, the recovery form is even, the ovarian cumulus-ovocyte-complex body (COCs) of compact structure, with twice of ripe liquid (M199+10%FBS+0.01U/mLbFSH+0.01U/mL bLH+1 μ g/mL estradiol) washing, then ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid with 50-60 piece/hole, at 38.5 ℃, 5%CO
2After maturation is cultivated 18-20h in the incubator, after sophisticated ovocyte being put into the pipe vibration 2-3min of 0.1% Unidasa, blow and beat gently with Glass tubing again, cumulus cell and ovocyte are broken away from fully, select complete form, tenuigenin evenly and the ovocyte of discharging first polar body be cytosol receptor.
3. the vitro culture of nuclear transfer procedure and clone embryos
The ovocyte that will have first polar body moves into and contains in the operation liquid of M199+10%FBS+7.5 μ g/mL cytochalasin B, under 200 power microscopes, above polar body, zona pellucida cut an osculum with glass needle, again with internal diameter be 20 μ m Glass tubing with first polar body with and the ovocyte of below in karyomit(e) absorb in the lump, the solution of putting into M199+20%FBS again give a baby a bath on the third day after its birth all over after, place incubator standby.
With serum starvation 2-4 days for nucleome clones with 0.25% trypsin trypsin) digest 2-4min, is that the donorcells of 10-12 μ m moves in the ovocyte zona pellucida removed nuclear with 20 μ m diameter Glass tubings with diameter, put it into zimmerman then and merge in the liquid or 0.3M N.F,USP MANNITOL (Mannitol), 0.15mmol/L Ca
2+, 0.15mmol/L Mg
2+Put into integration slot in the liquid behind the 3-5min, rotating ovocyte makes for the nucleome cell vertical with electric field with the ovocyte contact surface, be 2.5kV/cm, burst length to be that 10 μ s, pulse number are after 2 times, recurrent interval are to merge (fusion instrument is the ECM-2001 of BTX company) under the condition of 1s simultaneously in the field intensity of DC pulse, move in the M199+10%FBS liquid rapidly, place 0.5h, its fusion rate is 50%-95%.Select reconstituted embryo and carry out external activation according to following two kinds of methods:
1) handles with B cytochalasin B and cycloheximide, the method of handling is for to handle reconstituted embryo 1 hour in the CRlaa+5%FBS liquid that contains 0.25ug/ml B cytochalasin B and 10ug/ml cycloheximide, be transferred to then and continue in the CRlaa+5%FBS liquid that contains the 10ug/ml cycloheximide to handle after 4 hours to move into again in the CRlaa+5%FBS liquid, at 38.5 ℃, 5%CO
2Cultivate in the incubator, observe embryo's development condition respectively behind 2d and 7d, the result shows the spilting of an egg rate 40%-85% of 2d, develops into clone's blastaea in 7 days that continue to cultivate after 5 days, and clone's blastocyst rate is 20%-60%.
2) carry out combination treatment with electricity irritation and B cytochalasin B and cycloheximide, the method of handling is for to move into reconstituted embryo in the CRlaa+5%FBS liquid, in field intensity is to handle 10-40 μ s in the DC pulse field of 2.0kV/cm, and then this reconstituted embryo handled 1 hour in the CRlaa+5%FBS liquid that contains 0.25ug/ml B cytochalasin B and 10ug/ml cycloheximide, be transferred to then and continue in the CRlaa+5%FBS liquid that contains the 10ug/ml cycloheximide to handle 4 hours.Observe embryo's development condition respectively behind 2d and 7d, the result shows the spilting of an egg rate 45%-90% of 2d, develops into clone's blastaea in 7 days that continue to cultivate after 5 days, and clone's blastocyst rate is 30%-65%.
4, embryo transfer and gestation detect
7d bull that form is good clone blastaea moves in the horn of uterus of the recipient cattle of the same period.30d after transplanting carries out B ultrasonic to receptor cow and detects determining the situation of being impregnated, and the 60d after transplanting and 90d carry out rectum and detect to determine pregnancy rate respectively, and the result shows conception rate 60%, and pregnancy rate is 40%.
5, to cow in calf routinely method for breeding raise, through 280 days, cow in calf normal labor obtained the somatic cell clone breeding oxen.
Claims (8)
1, a kind of method of breeding breeding oxen may further comprise the steps:
1) sets up for nucleome clone with the somatocyte of breeding oxen, and carry out the same period and handle;
2) with merging through moving in the non-nucleus egg mother cell of the processing same period in the step 1), obtain reconstituted embryo for the nucleome cell;
3) with step 2) in the reconstituted embryo that obtains in external activation and cultivated 2-7 days, obtain blastaea, described blastaea is moved in the recipient cattle horn of uterus of estrus synchronization, obtain the somatic cell clone breeding oxen.
2, method according to claim 1 is characterized in that: described is breeding oxen body skin fibroblast line for the nucleome cell.
3, method according to claim 1 and 2 is characterized in that: be treated to the described same period described nucleome clone serum starvation 2-4 days that supply.
4, method according to claim 1 is characterized in that: step 2) in, describedly be fused to electricity irritation and merge.
5, method according to claim 4 is characterized in that: it is 2.0-2.5kV/cm that described electricity irritation is merged in field intensity, and the burst length is 10-40 μ s, and pulse number is 1-3 time, and the recurrent interval is to carry out in the DC pulse field of 1s.
6, method according to claim 1, it is characterized in that: in the step 3), described external activation adopts B cytochalasin B and cycloheximide to handle, described treatment process is transferred to then and continues in the nutrient solution that contains the 10ug/ml cycloheximide to handle 4 hours for described reconstituted embryo was handled 1 hour in the nutrient solution that contains 0.25ug/ml B cytochalasin B and 10ug/ml cycloheximide.
7, method according to claim 1, it is characterized in that: in the step 3), described external activation adopts electricity irritation and B cytochalasin B and cycloheximide to unite activation, described associating activated method is for earlier handling 10-40 μ s with described reconstituted embryo in field intensity is the DC pulse field of 2.0kV/cm, and then described reconstituted embryo handled 1 hour in the nutrient solution that contains 2.5ug/ml B cytochalasin B and 10ug/ml cycloheximide, be transferred to then and continue in the nutrient solution that contains the 10ug/ml cycloheximide to handle 4 hours.
8, according to claim 6 or 7 described methods, it is characterized in that: described B cytochalasin B is cytochalasin B or B cytochalasin B D.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410049790 CN1715401A (en) | 2004-06-29 | 2004-06-29 | Method for breeding herd bull |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410049790 CN1715401A (en) | 2004-06-29 | 2004-06-29 | Method for breeding herd bull |
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| CN1715401A true CN1715401A (en) | 2006-01-04 |
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| CN 200410049790 Pending CN1715401A (en) | 2004-06-29 | 2004-06-29 | Method for breeding herd bull |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104994728A (en) * | 2012-10-19 | 2015-10-21 | 移卵基因有限公司 | Method for generating genetically superior animals |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104994728A (en) * | 2012-10-19 | 2015-10-21 | 移卵基因有限公司 | Method for generating genetically superior animals |
| CN104994728B (en) * | 2012-10-19 | 2019-02-15 | 移卵基因有限公司 | Method for generating genetically good animal |
| US10927411B2 (en) | 2012-10-19 | 2021-02-23 | Trans Ova Genetics, L.C. | Methods for generating animals with desirable traits |
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