CN1304445A - Method for producing cloned tigers by employing inter-species nuclear transplantation technique - Google Patents

Abstract

The present invention provides a method for producing cloned tigers by employing inter-species nuclear transplantation technique. The method for producing cloned tigers of the invention comprises the steps of: preparing donor somatic cell lines collected from tiger; maturing oocytes collected from ovary of cow or cat in vitro; removing the cumulus cells surrounding the oocytes; cutting a portion of zona pellucida of the matured oocytes to make a slit, and squeezing out a portion of cytoplasm including the first polar body through the slit to give enucleated recipient oocytes; transferring a nucleus to the recipient oocyte by injection of the donor cells to the enucleated recipient oocytes, followed by the subsequent electrofusion and activation of the electrofused cells to give embryos; postactivating and culturing the embryos in vitro; and, transferring the cultured embryos into surrogate mothers to produce cloned tigers. The present invention can be widely applied in preservation of genetic characters of rare or endangered species permanently in the form of nuclear transferred embryos.

Description

A kind of by moving the method that technology is produced the clone tiger with consideration convey between planting

Background technology of the present invention

Invention field

The present invention relates to a kind of method of moving technology production clone tiger with consideration convey between planting; more particularly; relate to a kind of production with the technology of moving of consideration convey between planting and clone brave method, this method will be transferred to the ovocyte that comes from ox or cat from the somatic karyon that the tiger tissue obtains.The invention still further relates to the clone tiger embryo and the clone tiger that produce by aforesaid method.

Background of invention

People consider to produce animal with fertilization for a long time, and this relates to female and male gametophyte.Yet people have carried out huge effort produces the cloned animal with identical appearance and identical hereditary feature.

Over 30 years, may in Amphibians, clone zygote for known, but the filial generation (seeing: McGrath and Solter, science, 220:1300-1302,1983) that successfully in mouse, produces the clone by the pronucleus of replacing unicellular zygote at present.Though this first success has been arranged on cloned animal, the industrial same success of animal (is seen: Wakayama etc., nature, 394:369-374,1998) but report phase that night, because produce the clone mouse several problems are arranged, for example the minimizing of reprogramming etc. with the mature oocyte behind the 2-cell stage and zygote blastomere.

Relevant moving by consideration convey produced the clone livestock, and the first report is by doing the filial generation (seeing: Wiladsen, nature, 320:63-65,1986) that donorcells produces sheep with 8-to the blastomere of 16-cell zygote.After this, it is believed that to move to clone to have totipotent zygote blastomere, take this that cell can be divided into that each is unicellular by consideration convey.Yet, through the research work that continues, the first clone sheep produces by importing somatic cell nuclear and (sees: Wilmut etc., nature, 385:810-813,1997), thereby corrected growth theory formerly, and made and (see: Well etc., Reprod.Fertil.And Develop. producing clened cows, 10:369-378,1998) and the pig aspect report of successful example has appearred.

Produce embryo and the technology that produces the embryo by the somatocyte that discovers and uses existing wildlife, be it is believed that for the hereditary feature aspect of for good and all preserving rare endangered species be of great value.But, the also not successfully report of the existing wildlife of clone.It is found that, when these wildlifes of clone, if clone's animal is rare and is the shielded acceptor ovocyte that just is difficult to obtain.Therefore the technology that consideration convey moved between its cloned animal was just planted by application produces, and ovocyte obtains from the species that are closely related.The technology that this all consideration convey moves has formerly had openly (sees: Domink etc., Bio1.Repro., 60 (6): 1496-1502 (1999).But, this article at be to use the livestock that has carried out many research work, produce on the wildlife so still make it to be difficult to this technology is applied to.

In the case, the intensive reason is arranged, explore and develop a kind of improved method, move the wildlife that produces the clone with the somatocyte of wildlife by consideration convey between planting.

Summary of the invention

According to the present invention, we find: can produce the embryo of clone tiger and by described embryo's output clone tiger by the technology of moving of consideration convey between planting, the consideration convey technology of moving relates to the ovocyte that obtains from ox or cat with the somatocyte fusion of tiger between kind.

Therefore primary and foremost purpose of the present invention provides a kind of method of moving technology production clone tiger by consideration convey between planting.

Another object of the present invention is to provide the clone brave embryo by described method.

Another purpose of the present invention provides by what described method was produced and clones tiger by the somatocyte deutero-.

Brief description of drawings

Can know understanding above-mentioned purpose of the present invention and other purpose and feature of the present invention in conjunction with the accompanying drawings by following description, among the figure:

Fig. 1 is the somatic photo of donor.

Fig. 2 is a photo, the process that expression is held fixed (thin glass) pipe with one and a undercut cuts (carefully glass) pipe cutting acceptor ovocyte zona pellucida.

Fig. 3 is a photo, and expression is by taking the process that first polar body and karyon carry out stoning away from the acceptor ovocyte.

Fig. 4 is a photo, and expression is held fixed tube and syringe the process of somatocyte immigration non-nucleus egg mother cell with one.

Detailed description of the present invention

The method that the present invention produces the clone tiger comprises following steps: preparation from the donor body cell that tiger gathers with it is; Externally will cultivate ripe from the egg mother cell that ox or cat ovary gather; Remove egg mother cell mound cell on every side, the part of the egg mother cell oolemma that cutting is ripe and the kytoplasm that extruding part comprises first polar body are to obtain stoning acceptor egg mother cell; To examine immigration acceptor egg mother cell by donorcells being injected into enucleation oocyte, then obtain the embryo by electricity fusion subsequently and the cell that activates after electricity merges; Rear activation and extracorporeal culturing embryo; Then it is young to produce the clone tiger embryo who cultivates to be moved into the replace-conceive dam.

The method of production clone tiger of the present invention further specifies as follows. Step 1: preparation donorcells

The body cell system that gathers from tiger is prepared into donorcells: make donorcells although gather what cell from tiger without limits with it, but preferred clone comprises: from the cell of uterine irrigation liquid, endometrium, fallopian tubal, ear or muscle, mound cell or fetal fibroblast collection, can use conventional known method to change a little these clones of rear preparation (Mather﹠ Barnes, cell biology method, the 57th volume, Zooblast cultivation method, Academic Press, 1998).

For example, contain 1% penicillin-streptomysin (Gibco by in uterine irrigation liquid, adding; Every milliliter of 10000 unit penicillin, every milliliter of 10 milligrams of streptomysins) salt solution (PBS) of phosphate buffered is then centrifugal. The cell that gathers from uterine irrigation liquid is in the improved EagleShi medium of DulbeccoShi (DMEM) that has replenished nonessential amino acid, 10% hyclone (FBS) and 1% penicillin-streptomysin, at 39 ℃, 5% CO₂Environment under cultivate.

From the uterine epithelial cell of uterine endometrium or uterine tube collection with described PBS cleaning, trypsin treatment, under environment same as described above, cultivate then.

For the mound cell, use Unidasa solution-treated mound cell-ovocyte complex body to separate ovocyte mound cell on every side.With the mound cell at 39 ℃, 5% CO ₂Environment under make trypsin treatment and cultivated in above-mentioned similar mode more later in 30 to 60 minutes.

About ear inoblast and tire tiger inoblast, they obtain from the tissue that the inboard that wraps in the cortex on the cartilaginous tissue and tire tiger limb and body are collected respectively: under aseptic condition, clean and broken these tissues, then with trypsinase and type enzyme at 39 ℃, 5% CO ₂Environment handle down.These cells are also cultivated in the mode that is similar to above-mentioned donor somatocyte system.

Somatocyte system is cultivated by the cultivation of going down to posterity, serum starvation or freezing method stores.Going down to posterity of donorcells system cultivated termly by replacing old substratum to carry out with new substratum after trypsin treatment.Serum starvation cultivate by use the DMEM that replenishes 0.5%FBS with the method for Wilmut etc. carry out (see: Wilmut etc., nature, 385:810-813.1997).The clone of Chu Cuning is used as donorcells in the step of back like this.Step 2: preparation acceptor ovocyte

To cultivate ripe from the jejune ovocyte that the ovary of ox or cat is gathered external: from containing 10mM N-hydroxyethyl piperazine-N ＇-[jejune ovocyte of selection in the ovary the TCM199 cleaning medium of 2-ethylsulfonic acid (HEPES); then, depend on cultivate in the different developing medium that do not coexist of the animal of obtaining ovocyte ripe.The ovocyte that is obtained by ox makes its maturation (seeing Table 1) with the cultivation of TCM199-1 developing medium.The ovocyte that is obtained by cat makes its maturation (seeing Table 2) with the TCM199-2 developing medium cultivation that has replenished hCG (HCG).Either way at 39 ℃, 5% CO ₂Environment cultivated 16 to 22 hours down.

Table 1:TCM199-1 developing medium

Composition	Concentration
		TCM liquid	Gibco11150-059
Sodium.alpha.-ketopropionate	1mM
		Penicillin-Streptomycin sulphate	1% (penicillin 10000 units per ml, Streptomycin sulphate 10 mg/ml)

Table 2:TCM199-2 developing medium

Composition	Concentration
		TCM liquid	Gibco11150-059
Glutamine	2mM
		Sodium.alpha.-ketopropionate	0.36mM
Sodium.alpha.-hydroxypropionate	0.78mM
		Gentamicin	50μg/ml
HCG (HCG)	100IU/ml
		?BSA	?3g/l

Step 3: the stoning of acceptor ovocyte

Remove after the partially transparent band of mound cell around the sophisticated recipient cell and excision ovocyte, from ovocyte, remove the kytoplasm that part comprises first polar body, to obtain non-nucleus egg mother cell: at first strip off pipe and from the TCM199 cleaning medium that contains Unidasa, physically remove sophisticated ovocyte mound cell on every side with one.Clean the ovocyte stripped off with the TCM199 cleaning medium then, and they are moved into cytochalasin B solution.For cell stoning to stripping off, the part of the zona pellucida of the ovocyte that has stripped off with undercut pipe cutting puncture, to obtain a breach, from then on breach can comprise that 10% to 15% kytoplasm of first polar body extrudes from ovocyte to a part.Clean non-nucleus egg mother cell, and in the TCM199 developing medium, be incubated.Described cytochalasin B solution is to prepare by the cytochalasin B that is dissolved among the DMSO (dimethyl sulfoxide (DMSO)) with the dilution of TCM199 developing medium.Step 4: electricity merges the cell that donorcells and acceptor ovocyte and activation electricity merge

Donorcells is moved in the acceptor ovocyte, then carry out the electric cell that merges and activate the electricity fusion: before donorcells is injected into the acceptor ovocyte, move on in PHA-P (phytohaemagglutinin) solution with TCM199 developing medium cleaning non-nucleus egg mother cell and them.By donorcells is expelled in the breach that causes on the zona pellucida of the non-nucleus egg mother cell in the PHA-P solution, donorcells is moved on in the non-nucleus egg mother cell then.

Making electricity consumption cell manipulation device (BTX ECM2001) carry out electricity merges.Be placed in the chamber of two electrodes being placed on the embryo who replenishes with the reconstruction in the mannitol solution of TCM199 scavenging solution, the every side of these two electrodes in described chamber has one.The embryo being placed in the described chamber with its donorcells, described chamber is filled with mannitol solution facing to before the negative electrode.After twice DC pulse of 0.75 to 2.00 kv/cm of 1 second each 15 microsecond carried out the electricity fusion to the embryo at interval, clean the embryo that electricity merges with mannitol solution and TCM199 cleaning medium, in cytochalasin B, cultivate, and activation.If electricity merges and carries out in the N.F,USP MANNITOL medium of calcium ions, electricity merges and activation takes place simultaneously.Not so, activation is carried out after electricity merges.When in electricity merges N.F,USP MANNITOL medium in calcium ions not, carrying out, activate step and undertaken by insulation embryo in the dark in ionomycin solution.Remove ionomycin by cleaning the embryo then with the TCM199 cleaning medium that contains FBS or BSA.Described ionomycin solution is by being dissolved in the ionomycin preparation among the DMSO with the TCM199 cleaning medium dilution that contains BSA.Step 5: embryo's back activation and vitro culture

Embryo's back activation and vitro culture: activate by insulation back in cycloheximide solution or 4-dimethylamino-purine (DAMP) solution and to contain the activated embryo who is incubated in the TCM199 cleaning medium of FBS or BSA, and at 5% CO ₂Or at 5%CO ₂, 7%O ₂And 88%N ₂Mixed-gas environment under external cultivation.Described cycloheximide solution or DAMP solution prepare by add the cycloheximide or the DAMP that are dissolved in the ethanol in the medium of vitro culture.The medium of vitro culture comprises mTALP (seeing Table 3), mSOF (seeing Table 4) and mCR2aa (seeing Table 5) medium, and they all contain NaCl, KCl, NaHCO ₃, NaH ₂PO ₄, CaCl ₂, Sodium.alpha.-hydroxypropionate, glucose, phenol red, BSA, kantlex, indispensable amino acid, non-essential amino acid and L-glutaminate.

Selectively, use after the embryo of freezing preservation vitro culture is equipped with, and when planning to use, thaw.For frozen embryo, clean it with the PBS that contains FBS, and put into and contain penicillin-Streptomycin sulphate, CaCl ₂, glucose, MgCl ₂, Sodium.alpha.-ketopropionate and PBS refrigerant in.Slowly freezing the embryo in the refrigerant then, follow quick freezing in liquid nitrogen.When embryos frozen is taken out from liquid nitrogen and is thawed, they are placed about 5 seconds in air, in warm water, thaw then.In order from the embryo of thawing, to remove refrigerant, they are in turn put into from containing high density glycerine to the serial medium of the glycerine that contains lower concentration.

Table 3:mTALP medium

Composition	Concentration
		NaCl	?93.1-103.4mM
KCl	?3.1mM
		NaHCO 3	?25mM
NaH 2PO 4	?0.36mM
		Sodium.alpha.-hydroxypropionate	?15mM
CaCl 2.2H 2O	?1.7mM
		MgCl 2.6H 2O	?0.5mM
Sodium.alpha.-ketopropionate	?0.45mM
		Glucose	?1.5mM
Phenol red	?10μg/1
		?BSA	?8mg/ml
Kantlex	?0.75μg/ml
		EAA (indispensable amino acid)	?2％
NEAA (non-essential amino acid)	?1％
		L-glutaminate	?1mM
ITS (Regular Insulin-transferrin-Sodium Selenite substratum adds)	?0.5％

Table 4:mSOF medium

Composition	Concentration
		NaCl	99.1-106mM
KCl	7.2mM
		NaHCO 3	25mM
NaH 2PO 4	1.2mM
		Sodium.alpha.-hydroxypropionate	5mM
CaCl 2.2H 2O	1.7mM
		MgCl 2.6H 2O	0.5mM
Sodium.alpha.-ketopropionate	0.3mM
		Glucose	1.5mM
Phenol red	10μg/l
		?BSA	8mg/ml
Kantlex	0.75μg/ml
		EAA (indispensable amino acid)	2％
NEAA (non-essential amino acid)	1％
		L-glutaminate	1mM
?ITS	0.5％

Table 5:mCR2aa medium

Composition	First substratum (1-4 days)	The 2nd substratum (after 4 days)	Cleaning medium
				????NaCl	????114mM	????114mM	????114mM
????KCl	????3.1mM	????3.1mM	????3.1mM
				????NaHCO 3	????25mM	????25mM	????2mM
????NaH 2PO 4	????0.35mM	????0.35mM	????0.34mM
				Sodium.alpha.-hydroxypropionate	????15mM	????15mM	????15mM
??CaCl 2.2H 2O	????2mM	?????-	????2mM
				??MgCl 2.6H 2O	????0.5mM	????0.5mM	????0.5mM
????EAA	?????-	????1％	?????-
				????NEAA	????1％	????1％	????1％
Regular Insulin	????1％	????1％	????1％
				Glutamine	??????-	????1mM	????1mM
Padil	????0.37mM	???0.37mM	???0.37mM
				Citric acid	????0.33mM	???0.33mM	???0.33mM
??HEPES	??????-	?????-	???10.5mM
				Sodium.alpha.-ketopropionate	????0.3mM	????0.3mM	??????-
Glucose	??????-	????1.5mM	??????-
				Phenol red	????10μg/l	????10μg/l	????10μg/l
????BSA	????3mg/ml	??????-	????3mg/ml
				????FBS	??????-	????10％	???????-
Kantlex	????0.75μg/ml	????0.75μg/ml	????0.75μg/ml

Step 6: produce the clone tiger

The embryo of vitro culture is moved into the replace-conceive dam to produce tiger: will implant the uterus of replace-conceive dam the embryo in containing the PBS of FBS.

Based on above-mentioned method, the present invention has prepared the embryo by making karyon donor and acceptor ovocyte with ear cell of Korea S tiger and the ovocyte of Korea S ox respectively, i.e. SNU5 (Korea S tiger NT embryo).This embryo on March 10th, 2000 in the international preservation KCTC of mechanism (Korea S typical case culture collection center, Korea S KRIBB#52, OUNDONG, YUSONG-KU, TAEJON, 305-33, Korea S) preservation, preserving number is KCTC0752BP.

Further specify the present invention with following each embodiment now, these embodiment should be as limiting the scope of the invention.Embodiment 1: preparation donorcells and acceptor ovocyte

In order to prepare donorcells, carry out disinfection wiping Mao Houyong ethanol around the tissue and trimethyl-glycine (betadine) tissue off to male adult brave have sharp ears.With disinfectant apparatus dermal tissue harvesting (1 to 2 square centimeter of area), contain the PBS (salt solution of phosphate buffered that contains 0.5% penicillin (10000 units per ml)-Streptomycin sulphate (10 mg/ml) in moving into, GibcoShi BRL, U.S. Life Technology) in 50 milliliters of test tube.Then from the skin histology of described collection with sterilization scissors and scalpel isolation of cartilage with contain the fur skin, the tissue that obtains serving as a contrast the skin inboard on cartilage is as donor.This tissue cleans with PBS, is crushed to 100 orders then.Then at 39 ℃, 5% CO ₂In the PBS of the type enzyme of EDTA that contains 0.25% trypsinase, 1mM and 1 mg/ml, will organize insulation 1 hour under the environment, after tissue is by enzymic digestion, centrifugal two minutes with 1500 rev/mins, be suspended in DMEM (the improved EagleShi substratum of DulbeccoShi that has replenished 10%FBS, 1%NEAA (non-essential amino acid) and 1% penicillin-Streptomycin sulphate then, GibcoShi BRL, U.S. LifeTechnology) in.Suspension is moved in the Tissue Culture Dish, at 39 ℃, 5%CO ₂Environment insulation down to obtain somatocyte is.After this cell carries out trypsin treatment in the solution of the EDTA that contains 0.25% trypsinase and 1mM, then cell count is adjusted to 2 * 10 ⁴Individual cells/ml is with five equilibrium cell in the Eppendorf pipe.

Fig. 1 represents to be separated into individual cells with the somatocyte as the karyon donor.

On the other hand, for obtaining the acceptor ovocyte, draw the ovarian follicle of about 2 to 6 millimeter of diameter from the ovary of Korea S ox with 10 milliliters of syringes that have the 18G syringe needle.Then, liquor folliculi is moved into the bottom be decorated with in 100 millimeters plates of (width is 1 centimetre between line) of glazing bar, screening has uniform kytoplasm and the ovocyte of the mound cellular layer of sufficient amount is arranged on every side then.The ovocyte of selecting cleans three times with 2 milliliters of TCM199 cleaning mediums (seeing Table 6) in 35 millimeters plates, then cleans once with TCM199-1 developing medium (table 1).In the TCM199 medium that contains 0.1% estradiol solution (table 7), 2.5% follicle stimulating hormone solution (table 8) and 10%FBS, cultivate at last, to obtain the acceptor ovocyte.

Table 6:TCM199 cleaning medium

Composition	Concentration
		The TCM powder	Gibco31100-027
?HEPES	?10mM
		?NaHCO 3	?2mM
?BSA	?0.5％W/V
		Penicillin-Streptomycin sulphate	1% (penicillin 10000 units per ml, Streptomycin sulphate 10 mg/ml)

Table 7: estradiol solution

Composition	Concentration
		Estradiol	5 milligrams
Ethanol	10 milliliters

Table 8: follicle stimulating hormone solution

Composition	Concentration
		Follicle stimulating hormone	2AU
The TCM199 developing medium	10 milliliters

Embodiment 2: somatic consideration convey moves

The acceptor ovocyte of preparation cleans once with the TCM199 cleaning medium among the embodiment 1, moves into 0.1% Unidasa (the U.S. Sigma Chemical Co.) solution that the Unidasa stoste (in the TCM199 cleaning medium 10 milligrams every milliliter) of TCM199 cleaning medium and 111 microlitres by 1 milliliter is mixed with then.After removing the mound cell from ovocyte under the 0.1% Unidasa environment, the ovocyte after stripping off is cleaned three times and in the TCM199 cleaning medium, be incubated.Then ovocyte is moved in cytochalasin B (the U.S. Sigma Chemical Co.) solution that the cytochalasin B stoste (7.5 milligrams every milliliter of DMSO) of 1 milliliter of TCM199 cleaning medium by containing 10%FBS and 1 microlitre is mixed with, an and part of cutting the zona pellucida of each ovocyte with a kind of micromanipulator, to obtain a breach, from then on breach can be extruded 10% to 15% kytoplasm from ovocyte, to obtain non-nucleus egg mother cell.The step of preparation non-nucleus egg mother cell is described more specifically as follows: scratch diskette is placed on the micromanipulator platform, is equipped with one and holds fixed tube on the left arm of micromanipulator, be equipped with a undercut pipe cutting on its right arm.Then, control fixed tube respectively and the cutting pipe is placed along the direction at 9 o'clock and 3 o'clock, and by place in the centre microtubule controller they regulate can be along all directions free movement.Further regulate two microtubules to such an extent that can not touch scratch diskette, and make them not contact the centre that scratch diskette and their tip are placed on micro pipette by above micro pipette, moving up and down.Wash a mouthful pipe (internal diameter is greater than 200 microns) with one then ovocyte is moved into cytochalasin B solution from the TCM199 cleaning medium.At first micromanipulator is focused on the ovocyte, and further focus on by moving up and down these two microtubules with its coarse adjustment button and fine tuning button.Ovocyte is placed and to be able to the 12 o'clock direction of its first polar body along ovocyte, control then fixed tube along the 9 o'clock direction of ovocyte place near ovocyte with by liquid feeding press fit ovocyte.Fig. 2 represents the process with the zona pellucida of holding fixed tube and cutting pipe cutting ovocyte.As shown in Figure 2, ovocyte cuts to the direction at 11 o'clock from the direction at 1 o'clock with cutting pipe (2), and SC does not damage the kytoplasm of ovocyte.After this apply hydraulic pressure with ovocyte (3) separately to holding fixed tube (1), will hold fixed tube then with the perverse cutting pipe that penetrates oolemma contact against first polar body top, to manage the cutting part zona pellucidas by rubbing two.The above-mentioned breach of making on ovocyte both had been used for stoning and also had been used for the donor injection.Fig. 3 represents to remove from ovocyte the process of first polar body and karyon.As shown in Figure 3, ovocyte (3) with the placement that is vertically oriented of its breach, is held and decided its underpart and move preventing with holding fixed tube (1), gently squeeze its top to obtain non-nucleus egg mother cell with cutting pipe (2) then.Non-nucleus egg mother cell cleans three times with the TCM199 cleaning medium, is incubated in the TCM199 developing medium then.

After this, with micromanipulator the good donorcells of prepared beforehand is moved into non-nucleus egg mother cell.The PHA-P solution of at first using 400 microlitre TCM199 scavenging solutions and 100 microlitre PHA-P (phytohaemagglutinin) stostes (0.5 mg/ml in the TCM199 scavenging solution) to be mixed with is done the injection droplet of one 4 microlitre in the centre of scratch diskette.Then, do the droplet of two donorcellses with the PBS that contains 1%FBS, one on the injection droplet on the same scratch diskette, one is at it down.On these droplets, after the coated mineral oil, scratch diskette is placed on the micromanipulator platform.

Replace being installed in cutting pipe on the micromanipulator with syringe.Non-nucleus egg mother cell cleans three times with the TCM199 cleaning medium, moves into the injection droplet then.Donorcells is sucked syringe move into the injection droplet then.Fig. 4 represents an individual cells is moved into the process of non-nucleus egg mother cell.As shown in Figure 4, non-nucleus egg mother cell is oriented to its breach and placed at 1 o'clock, holds calmly with holding fixed tube, is injected into donorcells with syringe and hydraulic pressure through described breach then, with the embryo who obtains rebuilding.The embryo is cleaned three times with the TCM199 cleaning medium, in the TCM199 cleaning medium, be incubated then.Embodiment 3: electricity merges and activates

The embryo who rebuilds makes electricity consumption cell manipulation device, and (U.S., BTX ECM2001) carry out electricity and merge, and then activate.In the embryo's of containing reconstruction TCM199 developing medium, add 15 microlitres with a cleaning with a mouthful pipe and contain 0.28M N.F,USP MANNITOL, 0.5mM HEPES (pH7.2), 0.1mMMgSO ₄Mannitol solution with 0.05%BSA.In described medium after the insulation 1 minute, the embryo is placed on replenishes to cultivate 1 minute in the mannitol solution of TCM199 scavenging solution, with cleaning the embryo is moved into mannitol solution at last with a mouth pipe.Charge the mannitol solution that replenishes with the TCM199 cleaning medium in the chamber (3.2 millimeters No. 453 chambers) of electricity cell manipulation device, then described embryo is placed in the described chamber with its donorcells facing to negative electrode.After twice DC pulse of 0.75 to 2.00 kv/cm of 1 second each 15 microsecond carried out the electricity fusion to the embryo at interval, the embryo is moved into the TCM199 scavenging solution by means of mannitol solution, and clean three times with the TCM199 scavenging solution.

For activating the embryo that electricity merges, the embryo to be cultivated 4 minutes in the dark in ionomycin (U.S. Sigma ChemicalCo.) solution, described solution is a kind of TCM199 scavenging solution that contains the BSA of 5 μ M ionomycins and 1%.Described ionomycin stoste is by the ionomycin preparation of 1 milligram of dissolving in 1.34 milliliters DMSO.The activated embryo contain replenish with 35 millimeters plates of the TCM199 cleaning medium of 10%FBS in insulation 5 minutes with from embryo's flush away ionomycin.Embodiment 4: the embryo that the back is activated and the vitro culture electricity merges

The activated embryo activates 4 hours in back in cycloheximide (the U.S. Sigma Chemical Co.) solution of 25 microlitres, described cycloheximide solution is by add cycloheximide stoste (10 mg/ml are in ethanol) in the medium mTALP of vitro culture (seeing Table 3), is that 10 mcg/ml prepare to ultimate density.Screen the embryo then, the embryo who selects at 39 ℃ of temperature, 5% CO ₂Environment was cultivated 7 days down.

During based on above-mentioned method, the inventor use respectively from the ear cells of Korea S tiger as donorcells, make recipient cell with the ovocyte of Korea S ox, produce a kind of embryo, i.e. SNU5 (the brave NT embryo of Korea S).This embryo on March 10th, 2000 in the international preservation KCTC of mechanism (Korea S typical case culture collection center, Korea S KRIBB#52, OUNDONG, YUSONG-KU, TAEJON, 305-33, Korea S) preservation, preserving number is KCTC0752BP.Embodiment 5: use the ovocyte production embryo who obtains from cat

The present invention produces and cultivates the embryo with embodiment 1 to 4 is described with quadrat method, and difference is uses the ovocyte that obtains from cat as the acceptor ovocyte.Embodiment 6: embryo freezing and thaw and implant

The embryo who produces among the freezing embodiment 4 and 5 is with prolonged preservation.At first distribution a kind of refrigerant (seeing Table 9 and 10) in 35 millimeters plates is regulated refrigerator and is maintained to-5 ℃.Be elected to be embryos frozen and clean with the PBS that contains 10%FBS, insulation is 20 minutes in refrigerant.Then, the embryo is drawn into the centre that makes the refrigerant that contains the embryo be in suction pipe in 0.25 milliliter the French suction pipe, and the both sides of suction pipe are air.After having sealed the two ends of suction pipe with a warmed-up pliers, it is put into refrigerator, kept 5 minutes at-5 ℃.With one precooling pliers kind is gone into quick-frozen in the liquid nitrogen then.After planting, freezing with-0.3 ℃/minute speed to-30 ℃, kept 10 minutes when reaching-30 ℃ in temperature.At last the embryo is stored in the liquid nitrogen container.

Table 9: freezing PBS

Composition	Concentration
		PBS(1×)	Gibco14190-144
Sodium.alpha.-ketopropionate	0.033Mm
		Glucose	0.15mM
?CaCl 2.2H 2O	0.171mM
		Penicillin-Streptomycin sulphate	1% (penicillin 10000 units per ml, Streptomycin sulphate 10 mg/ml)
MgCl 2.6H 2O	?0.049mM

Table 10: refrigerant

Composition	Concentration
		Freezing PBS (table 9)	2.25ml(45％)
Foetal calf serum (FBS)	2.25mM(45％)
		Glycerine	0.5ml(10％)

For embryos frozen is thawed, preparation contains the medium that thaws that replenishes with the PBS of 20% FBS in 35 millimeters plates, and adds the thaw medium (see Table 9 and 11) of glycerine so that 0%, 3% and 6% glycerine respectively to be arranged.From liquid nitrogen, take out the refrigerated suction pipe then, in air, kept for 5 seconds, in the container that contains warm water (30 ℃) (diameter is greater than 20 centimetres), thaw then.After thawing, the gas cloud place at its two ends cuts open suction pipe, collects the medium that contains the embryo then.The test under microscope embryo.In order to remove refrigerant, they one after the other respectively are incubated 5 minutes in the medium that thaws that contains 6% glycerine, 3% glycerine and 0% glycerine from the embryo.

Table 11: medium thaws

Composition	6% glycerine PBS	3% glycerine PBS	0% glycerine PBS
				????PBS	(table 9)	(table 9)	(table 9)
????BSA	????0.5％	????0.5％	????0.5％
				Glycerine	????6％	????3％	????0％
Sucrose	????0.3M	????0.3M	????0.3M

The embryo of thawing is put into the PBS that contains 20% FBS, suck suction pipe then.Then it is moved into the uterus of replace-conceive dam.Embodiment 7: the embryo who relatively uses different donorcellses

In order relatively to use the not embryo's of isoacceptor ovocyte preparation difference, the embryos of embodiment 4 and 5 preparations are moved into the uterus of replace-conceive dam, and compare with regard to and the following then: the oocyte number that electricity merges, electric fusion rate (%), division rate (%), 8-cell stage number (%), 16-cell stage number (%), 32-somatic embryo number (%), morula/blastocyst are grown the conceived number (seeing: table 12) after number (%), the embryo number that shifts and embryo shift.Morula/blastocyst is grown number (%) is represented the embryo number of just being grown by vitro culture in the total embryo number that is moved generation by consideration convey of implanting the last stage ratio.

Table 12: the comparison of cloning brave embryo

The acceptor ovocyte	The oocyte number that electricity merges	Electricity fusion rate (%)	Division rate (%)	8-cell stage number (%)	16-cell stage number (%)	32-somatic embryo number (%)	Morula/blastocyst is grown number (%)	The embryo number that shifts	Conceived number after the transfer
										Ox	??1388	??50.9	??73.0	?38.6	?6.9	??5.2	????0.8	????6
Cat	??1478	??49.4	??61.0	?225	?2.6	??0.7	????0.1	????1	????1

As shown in table 12, use developmental rate and the conceptual quotient height of the ovocyte of ox than the ovocyte of using cat.This can be interpreted as, and many researchs of developing on the ovocyte of ox help to set up the ideal consideration convey and move condition, and also few for the research of developing on the ovocyte of cat.

Previous studies show that the livestock that has carried out many researchs can be used for kind between consideration convey move.Yet none is successfully used to produce clone's filial generation.Interrelate with above-mentioned situation, therefore the present invention has obtained very big success on generation somatocyte deutero-cloned animal, produced somatocyte deutero-clone filial generation.

Represent clearly and illustrate, the invention provides and a kind ofly move the method that technology is produced the clone tiger that this technology relates to the ovocyte fusion of stopping cell and ox or cat tiger by consideration convey between planting as above.A kind of embryo of clone tiger and method of cultivating the clone tiger with described embryo of providing is provided.The method according to this invention, consideration convey moves and produces its clone embryos between can planting by application, for good and all preserves the hereditary feature of rare animals on the brink of extinction, as a kind of approach of preserving wildlife.Except preserving wildlife, also be expected to use method of the present invention and develop many related application that consideration convey moves between kind that relate to.

After reading above explanation, those skilled in the art can be to shown in this paper and the described various modifications of carrying out.These modifications also fall in the described scope of claims of the present invention.

The preservation of related microorganism and other biomaterial proves (PCT detailed rules and regulations 13 two)

A. the preservation of the 9th page of capable described related microorganism of 1-5 and other biomaterial proves in the specification sheets
		B. preservation proves that other preservation is marked on other paper
Depositary institution Korea S typical case's culture collection center
		Holding structure address (comprising postcode and country): Taejon, Korea
Preservation date: on March 10th, 2000	Preserving number: KCTC 0752BP
C. other proofs (as not applying for not filling out this shelves) can continue the paper in other
		D. the designated country of testifying (if proof be not used in all designated states)
		E. separately submit proof (as not applying for not filling out this shelves) to
Following listed proof delivers submission to international office (refering in particular to total proof, as preserving number)

Only be used to accept office	Only be used for international office
		This page or leaf is accepted for international application	This page or leaf is accepted for international office
Authorize official	Authorize official

The PCT/RO/134 table

Claims (14)

1. produce the brave method of clone for one kind, the method includes the steps of:

The donor somatocyte that (I) preparation is gathered on one's body from tiger is;

(II) is external will cultivate ripe from the ovocyte that the ovary of ox or cat is gathered;

(III) removes the mound cell around the ovocyte, and a part of cutting sophisticated ovocyte zona pellucida to be causing a breach, and from then on the breach extruding part comprises the kytoplasm of first polar body, to obtain stoning acceptor ovocyte;

(IV) will examine immigration acceptor ovocyte by donorcells being injected into stoning acceptor ovocyte, obtain the embryo by electricity fusion subsequently and the cell that activates after electricity merges then;

(V) back is activated and extracorporeal culturing embryo;

(VI) moves into the replace-conceive dam to the embryo who cultivates then and clones brave young with generation.

2. according to the brave method of production clone of claim 1, wherein the somatocyte system of step (I) preparation comprises the cell of gathering from uterine irrigation liquid, uterine endometrium, uterine tube, ear or muscle, mound cell or tire inoblast.

3. according to the brave method of production clone of claim 1, wherein said somatocyte is to cultivate or freezing preservation by the cultivation of going down to posterity, serum starvation.

4. according to the brave method of production clone of claim 1, wherein in the step (III), after handling, physically remove ovocyte mound cell on every side with stripping off pipe with Unidasa.

5. according to the brave method of production clone of claim 1, wherein in the step (III), the stoning of ovocyte is carried out through the following steps: cause a breach with micromanipulator cutting ovocyte on ovocyte; Ovocyte is vertically oriented with its breach and holds fixed tube with one and hold the bottom of deciding ovocyte and move to prevent it; To comprise that through described breach 10% to 15% kytoplasm of first polar body extrudes from ovocyte with the top of undercut pipe cutting extruding ovocyte.

6. according to the method for the production of claim 1 clone tiger, wherein to shift be breach by through causing on the zona pellucida of ovocyte to the karyon in the step (IV), the donorcells injection entered the acceptor ovocyte of stoning carries out.

7. according to the method for the production of claim 1 clone tiger, wherein the electricity in the step (IV) merges that twice DC pulse being 0.75 to 2.00 kv/cm that applies by 1 second each 15 microsecond at interval carry out.

8. according to the brave method of production clone of claim 1, wherein in the step (IV), in the medium of calcium ions, carry out, activate step and take place with electric the fusion simultaneously if electricity merges.

9. according to the method for the production of claim 1 clone tiger, wherein in the step (IV),, electricity carries out if merging in the medium of calcium ions not, and activate step and in ionomycin solution, carry out in the dark.

10. according to the brave method of production clone of claim 1, wherein the back activation in the step (V) is undertaken by cultivate the embryo in cycloheximide solution or DAMP (4-dimethylamino-purine) solution.

11. according to the brave method of production clone of claim 1, wherein the vitro culture in the step (V) is undertaken by cultivate back activated embryo in mTALP, mSOF or mCR2aa medium.

12. according to the brave method of production clone of claim 1, this method also comprises the embryo's step for future use that is stored in vitro culture in the step (V), this step is to contain penicillin-Streptomycin sulphate, CaCl ₂, glucose, MgCl ₂, Sodium.alpha.-ketopropionate and phosphate buffered the brinish refrigerant in carry out after the frozen embryo.

13. embryo, be SNU5 (Korea S tiger NT embryo, KCTC0752BP), it is that step (I) by claim 1 is to (V), the ear cells of using the Korea S tiger respectively is as the karyon donor, and the ovocyte of usefulness Korea S ox (Bos taurus coreanae) produces as the acceptor ovocyte.

14. a clone tiger, it is that (Korea S tiger NT embryo is KCTC0752BP) as Embryo Production with the described SNU5 of claim 13 for step (VI) by claim 1.