CN1304444A - Method for producing human cloned embryos by employing inter-species nuclear transplantation technique - Google Patents

Abstract

The present invention provides a method for producing human cloned embryos by employing inter-species nuclear transplantation technique. The method for producing human cloned embryos of the invention comprises the steps of: preparing donor somatic cell lines collected from human; maturing oocytes collected from ovary of cow 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; and, postactivating and culturing the embryos in vitro. The human cloned embryos of the invention can be employed to obtain the human embryonic stem cells, which may be widely applied in biological and medical fields.

Description

A kind of by produce human cloned embryo's method with the consideration convey technology of moving between planting

Background technology of the present invention

Invention field

The present invention relates to a kind of method of producing human cloned embryo with the consideration convey technology of moving between planting, more particularly, relate to a kind of with kind between the consideration convey technology of moving produce human cloned embryo's method, this method is transferred to the somatic karyon that obtains from human tissue the mature oocyte that derives from ox.The invention still further relates to the human cloned embryo who produces 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.

Because the range gene recombinant plant (seeing: Schweizer etc., PlantJournal, 20:541-552,1999) with desirable useful crop feature has successfully been produced in biotechnology and engineered development recently.Aspect animal, the successful example of many cloned animals is arranged, comprising: clone sheep (sees: Wilmut etc., nature, 385:810-813,1997), clened cows (is seen: Wells etc., Reprod.Fertil.And Develop., 10:369-378,1998) and clone mouse (see: Wakayama etc., nature, 394:369-374,1998).Because the hi-tech that is not based upon on the biotechnology just can not realize the production of cloned animal, people the technical development of this technology of assessment in association area as a standard.

Because the stem cell of animal has the potential that develops into each organ, impel people simultaneously by obtaining and cultivating them and study the mechanism that it is divided into various organs.When carrying out this research, importantly adopt to have identical tissue-specific material, to reduce the difference in many different researchs.Yet, obviously be not always to obtain having identical tissue-specific appropriate materials.Provide and have identical tissue-specific material though adopted somatic clone technology facility recently, also unsatisfactory for human tissue.

In the case, the intensive needs are arranged, develop a kind of obtaining and have identical tissue-specific people's embryonic stem cells.

Summary of the invention

According to the present invention, we find: can successfully produce people's embryonic stem cells class by the consideration convey technology of moving between planting, the consideration convey technology of moving relates to the ovocyte that merges ox and uses human skin cell between kind, external human cloned embryo culture is arrived morula/blastocyst stage then.

Therefore primary and foremost purpose of the present invention provides a kind of method of producing human cloned embryo by the consideration convey technology of moving between planting.

Another object of the present invention is to provide human cloned embryo by described method.

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 the zona pellucida of (carefully glass) pipe cutting acceptor ovocyte.

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 human cloned embryo comprises following steps: preparation from the donor body cell that human body gathers is; The external egg mother cell that will collect from the ox ovary is cultivated ripe; Remove egg mother cell mound cell on every side, the part of the oolemma of the egg mother cell that cutting is ripe and the kytoplasm that extruding part comprises first polar body are to obtain stoning acceptor egg mother cell; Nuclear is moved into the egg mother cell of acceptor by donorcells is injected into enucleation oocyte, then obtain the embryo by electricity fusion subsequently and the cell that activates after electricity merges; Rear activation and at extracorporeal culturing embryo.

The human cloned embryo's of production of the present invention method further specifies as follows. Step 1: preparation donorcells

Body cell system from the human body collection is prepared into donorcells: make donorcells although gather what cell from human body without limits, preferred clone comprises: Skin Cell or the fibroblast that gathers from neonatal umbilical cord. Preferred is the Skin Cell that separates from skin histology as somatic clone. After can using conventional known method to change a little, prepare described clone (Mather﹠Barnes, cell biology method, the 57th volume, Zooblast cultivation method, Academic Press, 1998).

For example, clean and pulverize the inoblast of skin cells or neonatal umbilical cord.Then, with trypsinase and type enzyme at 39 ℃, 5% CO ₂Environment handle down these cells, in the improved EagleShi medium of Dulbecco ' sShi (DMEM) that has replenished non-essential amino acid, 10% foetal calf serum (FBS) and 1% penicillin-Streptomycin sulphate, under environment same as described above, cultivate then.

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 is cultivated by using the DMEM that replenishes 0.5%FBS and is carried out (seeing: Wilmut etc., nature, 386:810-813,1997) with the method for Wilmut etc.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 ox ovary is gathered external: select jejune ovocyte in the ovary from contain 10mM N-hydroxyethyl piperazine-N '-[2-ethylsulfonic acid] TCM199 cleaning medium (HEPES), then in the TCM199 developing medium (containing Sodium.alpha.-ketopropionate penicillin-Streptomycin sulphate) that has replenished estradiol, FSH (follicle stimulating hormone) and FBS at 39 ℃, 5% CO ₂Environment cultivate down and made above-mentioned oocyte maturation in 16 to 22 hours.Step 3: the stoning of acceptor ovocyte

Remove after the partially transparent band of mound cell around the sophisticated acceptor ovocyte 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 clear area of the ovocyte that has stripped off with undercut pipe cutting puncture, to obtain a breach, from then on breach can be extruded 10% to 15% the kytoplasm that comprises first polar body from ovocyte.Clean non-nucleus egg mother cell, and in the TCM199 developing medium, cultivate.Described cytochalasin B solution is by be dissolved in cytochalasin B formulations prepared from solutions in DMSO (dimethyl sulfoxide (DMSO)) solution with TCM199 developing medium dilution.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.Being placed in the chamber of two electrodes 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 is being placed in the described chamber with its donorcells facing to before the negative electrode, described chamber is filled with mannitol solution.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 carry out vitro culture.Described cycloheximide solution or 4-dimethylamino-purine 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 1), mSOF (seeing Table 2) and mCR2aa (seeing Table 3) 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 thawed, they are placed in air in warm water, thaw then about 5 seconds.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 1: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/l
		?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 2: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 3: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

Based on above-mentioned method, the present invention as the karyon donor, has prepared embryo SNU6 (human somatocyte system) with human skin cell.This embryo on June 19th, 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 KCTC0805BP.

Further specify the present invention with following each embodiment now, these embodiment not will be understood that and limit the scope of the present invention.Embodiment 1: preparation donorcells and acceptor ovocyte

In order to prepare donorcells, the tissue that will gather from people's skin cleans with PBS (salt solution of phosphate buffered, GibcoShi BRL, U.S. Life Technology), 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 then the DMEM that replenished 10%FBS, 1%NEAA (non-essential amino acid) and 1% penicillin-Streptomycin sulphate (Dulbecco ' the improved EagleShi medium of sShi, GibcoShi BRL, U.S. LifeTechnology) in.Suspension is moved in the Tissue Culture Dish, at 39 ℃, 5%CO ₂Environment is cultivated down to obtain somatocyte system.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 the somatocyte of one karyon donor.

On the other hand, for obtaining the acceptor ovocyte, drawing diameter with 10 milliliters of syringes that have the 18G syringe needle from the ovary of Korea S ox is the ovarian follicle of 2 to 6 millimeter.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 4) in 35 millimeters plates, then cleans once with TCM199 developing medium (seeing Table 5).In the TCM199 developing medium that contains 0.1% estradiol solution (table 6), 2.5% follicle stimulating hormone solution (table 7) and 10%FBS, cultivate at last, to obtain the acceptor ovocyte.

Table 4:TCM199 cleaning medium

Composition	Concentration
		The TCM powder	Gibco?31100-027
?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 5:TCM199 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 6: estradiol solution

Composition	Concentration
		Estradiol	5 milligrams
Ethanol	10 milliliters

Table 7: 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, cultivate.Then in cytochalasin B (the U.S. Sigma Chemical Co.) solution that the cytochalasin B stoste (7.5 milligrams every milliliter of DMSO) that moves into 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 to 15% 10% of 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.Control fixed tube then 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 be placed on the centre of micro pipette by above micro pipette, moving up and down the tip that makes them.Wash a mouthful pipe (internal diameter is greater than 200 microns) with one then ovocyte is moved into cytochalasin B solution.At first micromanipulator is focused on the ovocyte 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 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 to separate ovocyte (3) to holding fixed tube (1), will hold fixed tube and the cutting pipe that pierces through zona pellucida against first polar body top then, to manage the cutting part zona pellucidas by two of frictions.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 this 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), 0.1mM MgSO 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 adding cycloheximide stoste (10 mg/ml are in ethanol) in vitro culture medium mTALP, preparing to ultimate density 10 mcg/ml.Screen the embryo then, the embryo who selects at 39 ℃ of temperature, 5% CO ₂Environment was cultivated 7 days down.In culturing process, monitor embryo's growth (seeing Table 8) by elapsed-time standards.

Table 8 according to kind between consideration convey move derive embryo's growth of the human skin cell that obtains.

Electricity merges oocyte number	Electricity fusion rate (%)	Division rate (%)	2-cell stage number (%)	8-somatic embryo number (%)	Mulberry embryo/blastocyst is grown number (%)
						??203	??46.8	??51.6	????51.6	????34.7	????5.3

As shown in table 8, make it can be by human cloned fetal development to mulberry/blastocyst stage for transfer techniques between it clearly illustrated that kind, and in fact it be convenient to the mulberry/blastocyst manufacturer embryonic stem cell-like by growth.

Based on above-described method, the present invention as the karyon donor, produces a kind of embryo with human skin cell, SNU6 (human somatocyte system).Described embryo on June 19th, 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 KCTC0805BP.Embodiment 5: embryo freezing and thaw and implant

With embryo cryopreservation 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, and put into refrigerant insulation 20 minutes with the PBS that contains 10%FBS.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×)	Gibco?14190-144
Sodium.alpha.-hydroxypropionate	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)	2.25ml(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	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

Clearly represent and illustrate as above, the invention provides a kind of method of producing human cloned embryo by the consideration convey technology of moving between planting, comprise handle and move on to from the ovocyte that the ox body obtains, and produce human cloned embryo with described method by human somatocyte deutero-consideration convey.The method according to this invention is produced human cloned embryo and can be used to obtain the hESC, and this hESC can be widely used for various purposes, as other application in disease treatment and the medicine and pharmacology.

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

The preservation of related microorganism and other biomaterial proves

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A. the preservation of the 8th page of capable described related microorganism of inverse 1-4 and other biomaterial proves in the specification sheets
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Depositary institution Korea S typical case culture collection center (KCTC)
		Holding structure address (comprising postcode and country): Taejon, Korea
Preservation date: on June 19th, 2000	Preserving number: KCTC 0805BP
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The PCT/RO/134 table

Claims (13)

1. method of producing human cloned embryo, the method includes the steps of:

The donor somatocyte that (I) preparation is gathered from human body is;

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

(III) removes the mound cell around the ovocyte, and a part of cutting sophisticated ovocyte zona pellucida is to produce breach and to comprise the kytoplasm of first polar body by this breach extruding part, to obtain stoning acceptor ovocyte;

(IV) moves into the acceptor ovocyte by donorcells being injected into stoning acceptor ovocyte with a nuclear, and obtains the embryo by electricity fusion subsequently and the cell that activates after electricity merges;

(V) back is activated and extracorporeal culturing embryo.

2. according to the human cloned embryo's of production of claim 1 method, the inoblast that the somatocyte of preparation system comprises human skin cell or picks up from neonatal umbilical cord in the step (I) wherein.

3. according to the human cloned embryo's of production of claim 1 method, wherein said somatocyte system is by cultivations of going down to posterity, serum starvation cultivation or freezing the preservation.

4. according to the human cloned embryo's of production of claim 1 method, wherein in the step (III), after Unidasa is handled, physically remove mound cell around the ovocyte with stripping off pipe.

5. according to the human cloned embryo's of production of claim 1 method, wherein in the step (III), the stoning of ovocyte is by with micromanipulator cutting ovocyte and cause a breach 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; With a undercut pipe cutting through this breach comprise that 10% to 15% kytoplasm of first polar body extrudes from ovocyte.

6. according to the human cloned embryo's of production of claim 1 method, 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 human cloned embryo's of production of claim 1 method, wherein electricity 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 in the step (IV).

8. according to the human cloned embryo's of production of claim 1 method, wherein in the step (IV), in the medium of calcium ions, carry out, activate step and electricity and merge simultaneously and take place if electricity merges.

9. according to the human cloned embryo's of production of claim 1 method, 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 human cloned embryo's of production of claim 1 method, wherein the back activation in the step (V) is to be undertaken by cultivate the embryo in cycloheximide solution or DAMP (4-dimethylamino-purine) solution.

11. according to the human cloned embryo's of production of claim 1 method, 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 human cloned embryo's of production of claim 1 method, wherein further comprise 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 phosphoric acid buffer the brinish refrigerant in carry out after freezing.

13. an embryo, SNU6 (human somatocyte is KCTC 0805BP), it is the method by claim 1, makes the karyon donor with human skin cell respectively, the ovocyte of Korea S ox is produced as the acceptor ovocyte.

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