CN100404675C - Production process of somatic cell clone pig - Google Patents
Production process of somatic cell clone pig Download PDFInfo
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- CN100404675C CN100404675C CNB2006100031912A CN200610003191A CN100404675C CN 100404675 C CN100404675 C CN 100404675C CN B2006100031912 A CNB2006100031912 A CN B2006100031912A CN 200610003191 A CN200610003191 A CN 200610003191A CN 100404675 C CN100404675 C CN 100404675C
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Abstract
The present invention discloses a production method of a somatic cell clone pig, which comprises the following steps: a pig fetus mechanocyte is used as a donor cell for nuclear transplantation, an oocyte of an in vitro mature sow before a puberty is used as a recipient cell for nuclear transplantation, the donor cell for nuclear transplantation is moved in the denucleated oocyte to construct a cloning embryo, and then, the cloning embryo is cultured in an in vitro mode and under the condition of the oxygen volume percentage content of 5 to 10% to obtain an early embryo; the early embryo is transplanted in a sow to be pregnant to gestate and produce the somatic cell clone pig. The method hugely reduces the production cost of the somatic cell clone pig and increases the production efficiency of the clone pig.
Description
Technical field
The present invention relates to a kind of method of producing somatic cell clone pig.
Background technology
The body-cell neucleus transplanting clone is meant and utilizes certain device and technique means, with somatocyte of animal and the Oocyte in Vitro reorganization embryo who removes the nucleus genetic material, and then reconstituted embryo is transplanted to the intrauterine for the treatment of pregnant parent in specific developmental stage, finish growth, produce and the process of somatocyte for homogeneity offspring in the nucleome heredity.Body-cell neucleus transplanting is produced clone pig and be may further comprise the steps: gather the somatocyte of pig, set up somatocyte system; The external ovocyte that will gather from pig ovary is cultivated ripe; Donorcells nuclear is moved in the non-nucleus egg mother cell; Electricity by subsequently merges and activation obtains the embryo then; Vitro culture treats that to blastaea or immigration pregnant sow is to produce clone pig.
The porcine somatic cell clone has very important using value, can enrich the means of local variety guarantor kind and meat quality, disease-resistant improvement on the agricultural; Medically can be for human xenotransplant, disease model and new medicament screen provide ideal material, so somatic cell clone pig has boundless application prospect.In view of the organ of pig is that people wish to utilize pig that the people is provided needed various organs, carry out xenotransplant (Xenotransplantation) in more approaching with the people all on the volume or on function.Somatic cell clone pig has huge application potential aspect the animal disease model of setting up the people and the human organ's transplanting, has become the focus of current somatic cell clone research.The somatic cell clone of pig is that difficulty is relatively large in the various domestic animals, and the maturation in vitro of porcine oocytes is second-rate, and the fetal development ability of vitro culture is very low, and will have 4 embryo qualities attached planting simultaneously at least, just can bring out enough pregnant signals.After somatic cell clone sheep " many jasmines " in 1997 success, up to 2000, somatic cell clone pig has just had breakthrough finally, and Polejaeva etc. have obtained the success of somatic cell clone pig first, and the same year, Onishi etc. successfully obtained somatic cell clone pig respectively in succession with Betthauser etc.Polejaeva etc. adopt two step of binary acceptor nucleus transplantation, make the mature egg of going out in G0 phase granulosa cell and the body form reconstructed embryo earlier by electric integration technology, carry out the nuclear transplantation second time with the protokaryon that forms as nuclear donor again, successfully obtained the first routine somatic cell clone pig (5 piglets).Onishi etc. have also reported and have utilized porcine fetus fibroblasts to make nuclear donor, by the donorcells nuclear substance of Piezo after with rupture of membranes be injected directly into cylinder mature, in the enucleation oocyte kytoplasm, obtained a clone piglet.Bettauser etc. utilizes the ovocyte of maturation in vitro subsequently, merges to activate with electricity through electricity to obtain reconstructed embryo, has improved, simplified the program of somatic cell clone pig from many aspects, has established the common technology of clone pig today.Since 2000, at several countries be born in succession somatic cell clone pig and transgenic pig, but its successful efficient often was lower than 1%.
After the clone pig success was captured, people utilized somatic cell clone technique to begin to explore production transgenosis, genomic modification (as gene targeting) pig again, and calendar year 2001, Park etc. have successfully obtained the pig of commentaries on classics green fluorescent protein (GFP) gene.It is nuclear donor with transgenosis boar skin flbroblast that Bondioli etc. have transplanted 299 pieces, and the ovum of cylinder mature is the nuclear transfer embryo of cytosol receptor, gives birth to 2 piglets.The multiparity sow ovum of maturation in vitro such as Lai in 2002, the scheme that adopts fusion/activation to finish has synchronously obtained the gene knockout somatic cell clone pig, it indicates and gene site-directedly knocks out technology and nuclear transfer technology successfully combining on pig, but its total efficiency also fails to surpass 1%.
So far, though the whole world has had more than ten study group to obtain porcine somatic cell clone's success, the efficient of clone pig generally is no more than 1%.As previously mentioned, the somatic cell clone pig preparation process comprises that donorcells system sets up and preparation, obtaining and maturation of ovocyte, nuclear transplantation donorcells and non-nucleus egg mother cell merge, the cultivation of clone embryos, clone embryo transplantation and treat that pregnant sow gestation produces son, each link all is vital for the success or not and the efficient height of clone pig.
The stand-by mode of donorcells generally is that the cell that goes down to posterity is cultivated in existing digestion before nuclear transplantation, once uses up, and does not continue next time to utilize.Put into 4 ℃ of standby clones of refrigerator cold-storage at the cell that ox, sheep have the people to attempt utilizing the cell of 4 ℃ of refrigerations promptly to digest for nuclear donor, can make full use of the cell of the difficult acquisitions such as cell, especially transgenosis of digestion, and enrich the stand-by mode of donorcells.
The acceptor ovocyte of clone pig is cylinder mature at first, and the cost of sophisticated ovocyte is very high in super row's modus operandi acquisition volume, and workload is very big; Ripe gradually along with the oocyte in vitro maturation technology, people begin to utilize the ovocyte of maturation in vitro, promptly gather the slaughterhouse and butcher sow depleted ovary, will dash the prematurity ovum got in the laboratory in the culture medium culturing maturation from the ovarian follicle of ovary.The research overwhelming majority that has obtained at present clone pig is to utilize the ovum of cylinder mature or the ovum of multiparity sow (having given birth to the sow of piggy) maturation in vitro, and these investigators find to come from present the multiparity sow by contrast ovum has better ectogenesis potentiality than the ovum of sow pig (the not sow of living piggy).But dash the ovum cost get cylinder mature very high (maturation in vitro tens times), the ovary cost of buying the multiparity sow also very high (be sow pig ten times), and often be difficult to obtain, if carry out scale operation, the preparation cost of somatic cell clone pig will be quite high.Nowadays the market pig of butchering on a large scale in the slaughterhouse generally all is the sow pig that is in 6 monthly ages before puberty, the ovary resource is very abundant, cost is lower, be suitable for the scale operation and the research of somatic cell clone pig, but the ovum quality of puberty father's former wife's pig ovary is relatively poor, often ovarian follicle is not of uniform size, and aspects such as tenuigenin maturation are not as multiparity sow or cylinder mature ovum.Therefore how setting up better ripe scheme, to make full use of the abundant puberty father's former wife's pig ovary resource in slaughterhouse be pendulum tempting and challenging subject in face of the investigator.
Porcine oocytes ectogenesis ability and ripe liquid and culture condition have close getting in touch.Present generally two kinds of basic maturation medium plinth liquid formula of usefulness: the NCSU-23 of no BSA and TCM-199.And PZM-3 is a kind of new embryo culture medium, and this substratum is that Japanese scholar uses (Yoshiokaet al.2002) first on pig produced in vitro embryo culture, and also having no talent so far is used for PZM-3 the trial of oocyte in vitro maturation aspect.
Compare with other animals, the outer developmental potency of pig embryoid body is lower, and the blastaea rate is low, and cell count is few (to be the interior embryo's of body 1/4 approximately.This has reflected that condition of in vitro culture departs from internal milieu, and one of them important factor is the oxygen partial pressure of gas phase condition.Usually people are to use 5%CO2, carry out vitro culture in the incubator of 95% air (about 20%O2), and the oxygen partial pressure in uterine tube and uterus are lower than air.Cultivate down the embryo at hyperoxia (20%O2), may produce more oxyradical and be harmful to embryo's growth.At present, cultivation pig embryo's substratum has multiple, and wherein NCSU-23 is the most frequently used embryo culture medium, also is to generally acknowledge embryo culture medium preferably at present.Recently according to people's the uterine tube and the embryo medium GIII of uterine luminal fluid development, be that the substratum that liquid is changed in a cultivation in two stages is G1.3/G2.3, can adapt to the different fetal development stages to energy (glucose) and amino acid whose needs, it can improve the quality (especially blastomere number) that multiple animal embryo is grown.The raising of cell count may be because it be a more complete substratum, and G1.3/G2.3 has the equilibrated nutritive ingredient, and the concentration of glucose is respectively 0.5mM and 3.15mM, contains pyruvic acid, lactic acid and the amino acid of suitable concentration and VITAMIN etc.
Summary of the invention
The purpose of this invention is to provide a kind of method of producing somatic cell clone pig.
The method of production somatic cell clone pig provided by the present invention, be to be the nuclear transplantation donorcells with the porcine fetus fibroblasts, puberty father's former wife's porcine oocytes with maturation in vitro is the nuclear transplantation recipient cell, the nuclear transplantation donorcells is moved into non-nucleus egg mother cell be built into clone embryos, described clone embryos is to carry out vitro culture under the gas phase condition of 5-10% to obtain body early embryo at the oxygen volumn concentration, described body early embryo is implanted into treats pregnant sow gestation and produce somatic cell clone pig.
Described porcine fetus fibroblasts is handled through following method: existing digestion existing with or digestion after 2-8 ℃ of refrigeration 2-24 hour again.
Puberty father's former wife's porcine oocytes of described maturation in vitro is at the NCSU-23 of no BSA or do not have to cultivate in the PZM-3 maturation culture solution of BSA and obtain with puberty father's former wife porcine oocytes.
The NCSU-23 of described no BSA or the PZM-3 maturation culture solution that does not have a BSA are that NCSU-23 or PZM-3 add 10% (volume ratio) pig follicle liquid (PFF), 10ng/ml Urogastron (EGF), 10IU/ml human chorionic gonadotrophin (hCG) and 10IU/ml pregnant mare serum gonadotrop(h)in (PMSG) (PMSG).
In the aforesaid method, described clone embryos is to carry out vitro culture under 7% the gas phase condition to obtain body early embryo at the oxygen volumn concentration.
Described gas phase condition is preferably and contains 7%O
2, 5%CO
2, 88%N
2Mixed gas, described percentage composition is a volumn concentration.
The used basic medium of described somatic cell clone embryo's vitro culture is NCSU-23, PZM-3 or GIII, is preferably NCSU-23.Wherein, the substratum of described somatic cell clone embryo's vitro culture is NCSU-23, PZM-3 or GIII in the somatic cell clone fetal development to the 2-4 cell stage; Behind the 2-4 of somatic cell clone fetal development cell stage and 2-4 cell stage NCSU-23, PZM-3 or the GIII that adds 1-10ng/ml EGF.
The substratum that described somatic cell clone embryo's vitro culture is used is NCSU-23 in the somatic cell clone fetal development to the 2-4 cell stage; Behind the 2-4 of somatic cell clone fetal development cell stage and 2-4 cell stage the NCSU-23 that adds 1-10ng/ml EGF.
The interpolation concentration of described EGF is 10ng/ml.
In order to set up low cost, to reach high efficiency somatic cell clone pig production system on a large scale, the present invention is in the preservation mode of nuclear transplantation donorcells, ovocyte choose and the links such as raising of maturation in vitro, clone's blastaea efficient on be optimized, successfully obtained somatic cell clone pig.Be embodied in:
1) the nuclear transplantation donorcells of the present invention's employing is a porcine fetus fibroblasts, stand-by mode for existing digestion existing with and digestion after 2-8 ℃ of refrigeration 2-24 hour again, the result show 4 ℃ of refrigerations 12-20 hour with now to digest used donorcells the same, can be used for the nuclear transplantation donor and can effectively support the early development of clone embryos;
2) the nuclear transplantation acceptor of the present invention's employing is the ovocyte of sow before puberty; The ovocyte of sow before puberty is carried out maturation in vitro cultivate, adopted traditional oocyte maturation liquid promptly not have the NCSU-23 of BSA, and a kind of new oocyte maturation liquid PZM-3, the result shows that PZM-3 can be used as a kind of new oocyte maturation liquid;
3) the present invention has carried out vitro culture research to the clone embryos that obtains, and the result shows under the gas phase condition with 20%O2 (5%CO2,95% air) and compares (7%O under hypoxia condition
2, 5%CO
2, 88%N
2Gas phase condition under) significantly improved the developmental potency of clone embryos, improved embryo's blastaea rate and cell count; The present invention also gropes the nutrient solution of clone embryos vitro culture, adopting three kinds of substratum altogether is that NCSU-23, PZM-3 or GIII cultivate, the result shows that three kinds of substratum can both be used to clone the vitro culture of blastaea, and wherein GIII has improved the cell count of blastaea widely; The 2-4 cell stage that the present invention simultaneously also grows at clone embryos adds 10ng/ml EGF in substratum, found that the blastaea rate that can significantly improve from clone's embryo of ovary ovocyte before puberty.
3 acceptor sows of co-transplantation of the present invention (multiparity) are transplanted back 30d and define preceding 2 gestation with the B ultrasonic detection, and pregnancy rate is 66.7% (2/3), is higher than mean level (ML) 30-50%.Treat that pregnant sow B790 pregnancy expires and gives birth to 3 piglets, the natality 1.3% (3/230) of this experiment clone pig has surpassed the difficulty efficient 1% of generally acknowledging.3 pigs of birth, 1 survival, very healthy always after the birth.The present invention has greatly reduced the production cost of somatic cell clone pig, and has improved clone pig production efficiency, has set up a whole set of and can be used for the effective ways of scale operation somatic cell clone pig.Also human transplant organ is transplanted research and disease model foundation is laid a good foundation in order to carry out in the foundation of somatic cell clone pig production system simultaneously.
The present invention utilize the porcine somatic cell of 4 ℃ of refrigerations in the world first and before puberty the ovocyte of gilt maturation in vitro make up clone embryos, under hypoxia condition, cultivate clone embryos, successfully produce somatic cell clone pig.The success of 4 ℃ of refrigeration donorcells clone pigs utilizes the transgenic cell that is difficult for obtaining to come clone pig that an effective way is provided for saving from now on.Puberty father's former wife's in-vitro maturity of porcine oocytes is second-rate, but is difficult to obtain on a large scale the ovum (market pig is generally butchered before puberty) of multiparity sow in experiment, and this is a big bottleneck of somatic cell clone pig industrialization.The present invention utilizes the ovocyte before puberty to carry out maturation in vitro and obtains the clone pig success, has broken through the restriction of ovum, for large-scale industrialized production clone pig has from now on been established solid basis.
Description of drawings
Fig. 1 is ovocyte-cumulus cell complex body photo
Fig. 2 is sophisticated ovocyte photo
The early stage clone embryos photo that Fig. 3 cultivates down for hypoxemia
Fig. 4 is a body-cell neucleus transplanting blastaea photo
Fig. 5 is a somatic cell clone blastaea Hoechst33342 cell counting photo
Fig. 6 is the back 7 days somatic cell clone pig photo of birth
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment 1, somatic cell nuclear transfer technique are produced the condition optimizing experiment of clone pig
Key instrument equipment among the embodiment:
Bechtop; Stereoscopic microscope; CO2gas incubator; Micromanipulation system; Inverted microscope; Draw the pin instrument; The broken needle instrument; The card grinding instrument; Stereoscopic hot platform; Be inverted hot platform; Fusion instrument; Just putting fluorescent microscope
Used various chemical reagent among the embodiment are except that indicating especially, all available from U.S. Sigma-Aldrich company.
Various solution preparations among the embodiment:
DMEM (high sugar is available from GIBCO) solution: the DMEM powder is dissolved in the 900ml Milli-Q ultrapure water, adds the NaHCO of 3.7g/L again by description of product requirement
3After, add the 0.11g/L Sodium.alpha.-ketopropionate more in addition, 3.57g/LHEPES, the Vetstrep of 66mg/L penicillin and 100mg/L is transferred pH to 7.2～7.4, is settled to 1L then.Use 0.20 μ m membrane filtration degerming then, be stored in after the packing 4 ℃ standby.Add the foetal calf serum FBS (available from Gibco/Hyclone) of 10%-20% before using, 1% non-essential amino acid (NEAA) constitutes the cell perfect medium.
No calcium magnesium DPBS liquid (DPBS-): get 0.20g/L KCl respectively, 0.20g/L KH
2PO
4, 8.00g/L NaCl, 2.16g/L Na
2HPO
47H
2O is dissolved in the 900ml Milli-Q ultrapure water, transfers PH to 7.2～7.4, is settled to 1L then.0.20 the degerming of μ m membrane filtration, or autoclaving, be stored in after the packing 4 ℃ standby.
0.25% (0.25g/100ml) trypsinase tissue digestion liquid: get 0.25g trypsinase Trypsin (1: 250) and 0.02g EDTA, be settled to behind the 100ml with 0.20 μ m membrane filtration degerming with DPBS-available from GIBCO, be stored in after the packing-20 ℃ standby.
Promptly added the tyrode's solution (PVA-TL-HEPES) of 0.1% (0.1g/100ml) polyvinyl alcohol (PVA) at the HEPES buffered towards ovum liquid: 0.1%PVA-TL-HEPES:
With 6.663g NaCl, 0.237g KCl, 0.168g NaHCO
3, 0.041g NaH
2PO
4, 1.868mlNa Lactate, 0.102g MgCi
2.6H
2O, 2.383g Hepes, 0.065g penicillin G, 0.05gstreptomycin sulfate, 0.294g CaCl
22H
2O, 0.100g polyvinyl alcohol (PVA), 2.186g sorbitol and 0.022g sodium pyruvate are dissolved in 700ml Milli-Q H
2Among the O.Use 100ml Milli-Q ultrapure water heating for dissolving PVA again, treat the PVA dissolving and be cooled to room temperature, join the 700ml Milli-Q H that contains mentioned component
2In the solution of O, transfer pH to use Milli-Q H behind the 7.2-7.4
2O is settled to 1000ml, and making osmotic pressure is 295-310mOsm.0.22 μ m filter filters packing, is put in 4 ℃ of preservations, deposits and is no more than for 3 weeks.Be used for dashing and get cumulus cell-ovocyte complex body (COCs).
DPBS-PVA is towards ovum liquid: melt 1g PVA with the heating of 100ml Milli-Q water earlier, get 9.55gDPBS (available from Gibico) powder after the cooling again, add Milli-Q water, make it to dissolve, and be settled to 1L.Be used to wash COCs and preparation and take off ovarian cumulus liquid.
NCSU-23 (North Carolina State University-23): get 300ml Milli-Q water earlier, be sequentially added into 3.178g NaCl again, 1.053g NaHCO3,0.178g KCl, 0.081g KH
2PO
4, 0.147g MgSO
47H
2O, 0.125g CaCl
22H
2O, 0.500g glucose, 0.073g glutamine, 0.438gtaurine, 0.273g Hypotaurine, 0.033g penicillin G, 0.025g streptomycin.Transfer pH to use Milli-Q H behind the 7.2-7.4
2O is settled to 500ml, and osmotic pressure is 280～290mOsm.Be used for oocyte in vitro maturation basic medium or embryo culture medium.0.22 μ m filter filters packing, is put in 4 ℃ of preservations, uses up in 3 weeks.
Porcine zygote medium-3 (PZM-3): get 300ml Milli-Q water earlier, be sequentially added into 3.156g NaCl again, 1.053g NaHCO
3, 0.373g KCl, 0.024g KH
2PO
4, 0.049g MgSO
47H
2O, 0.308g Ca-lactate5H
2O, 0.011g Na-pyruvate, 0.073g L-glutamine, 0.273ghypotaurine, 10ml BME amino acid solution, 5ml MEM non-essential amino acidsolution, 0.033g penicillin G, 0.025g streptomycin.Transfer pH to use Milli-Q H behind the 7.2-7.4
2O is settled to 500ml, and osmotic pressure is 273～283mOsm.
Oocyte in vitro maturation culture solution: NCSU-23 or PZM-3 add 10% (volume ratio) pig follicle liquid (PFF), 10ng/ml Urogastron (EGF), 10IU/ml human chorionic gonadotrophin (hCG) and 10IU/ml pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) are called NCSU-23 or the PZM-3 of no BSA.
Take off ovarian cumulus liquid (0.1% Unidasa): the DPBS-PVA that gets the above-mentioned preparation of 50ml is towards ovum liquid, press 100mg/mL and add Unidasa, slowly stir, make it dissolving and be settled to 100ml towards ovum liquid with DPBS-PVA, 0.20 μ m syringe needle filter is at the super clean bench inner filtration, divide to install in the 1.5ml centrifuge tube ,-20 ℃ of preservations are standby.
Fusion/activation liquid: 0.25M (4.555g/100ml) Mannitol, 0.1mM CaCl
22H
2O (be made into 0.147g/100ml earlier, 100 * mother liquor is got the 1ml mother liquor again and diluted), 0.1mM MgCl
26H
2O (be made into 0.2033g/100ml earlier, 100 * mother liquor is got the 1ml mother liquor again and diluted), 0.5mM HEPES (be made into 1.192g/100ml earlier, 100 * mother liquor is got the 1ml mother liquor again and diluted), 0.01%PVA (0.01g/100ml) transfers pH to use Milli-Q H behind the 7.2-7.4
2O is settled to 100ml, and osmotic pressure is 253～268mOsm.0.20 μ m filter filters packing, 4 ℃ of preservations in the super clean bench.
Embryo operation liquid (Hepes-NCSU-23 (no calcium)): 0.76965g NaCl, 0.0168g NaHCO
3, 0.0356gKCl, 0.0162g KH
2PO
4, 0.0293g MgSO
47H
2O, 0.1g Glucose, 0.0146g Glutamine, 0.15012g Taurine, 0.2383g HEPES, 0.4g BSA (bovine serum albumin), 0.0065g PenicillinG, 0.005g streptomycin, Milli Q H
2The O dissolving is settled to 100ml.Mentioned component is sequentially added into mixing, and accent pH is 7.2-7.4, and osmotic pressure is 285-300mOsm, and 0.20 μ m filter filters packing, and 4 ℃ of preservations are standby.
Micrurgy liquid: Hepes-NCSU-23 (no calcium) (above-mentioned embryo operation liquid)+7.5 μ g/ml CB (cytochalasin B)
Embryo medium: NCSU-23+0.4%BSA; PZM-3+0.3%BSA; GIII (G1.3 and G2.3): available from the product of new visual field medical facilities company limited (Wuhan City) agency's Sweden VITROLIFE company.
1, the foundation of porcine fetus fibroblasts system and the preparation of donorcells
Adopt the tissue block inoculation method to set up fetal fibroblast system, conventional passage and freezing preservation to the Chinese experimental that is in the 33rd day Gestation period (breeding period be 0 day) with miniature pig (fragrant pig).Used substratum is DMEM.
The donorcells preparation method: 2-6 time the porcine fetus fibroblasts of waiting to go down to posterity grows to 90% when converging, with 0.25% trypsinase tissue digestion liquid digestion, centrifugal, add the resuspended cell precipitation of 200 μ L DMEM solution at last, directly provide as nuclear transplantation donorcells (on-the-spot digestion method) maybe with the cell of digestion again after 4 ℃ of refrigerators are preserved 12h as nuclear transplantation donorcells (4 ℃ of refrigerations), and the relatively spilting of an egg rate of the clone embryos that obtains of both methods and the difference of blastaea rate.The experimental technique that obtains blastaea by the nuclear transplantation donorcells see reference document (Zhang Yunhai etc.: utilize somatic cell nuclear transfer technique to produce the pig transgene clone embryo of expressing green fluorescent protein, Chinese science, C collects, 2005,35 (5): 439-445).The result is as shown in table 1, shows that on-the-spot digestion method and 4 ℃ of refrigerations of adopting the tryptic digestion centrifugal cell harvesting to do donor provide the nuclear transplantation donorcells, and the influence that two kinds of donorcells stand-by modes are grown clone embryos does not have significant difference (table 1).
The influence that table 1 donorcells stand-by mode is grown clone embryos
| Stand-by mode | Cultivate embryo number | Spilting of an egg number (spilting of an egg rate %) | Blastaea number (blastaea rate %) |
| On-the-spot digestion method | 181 | 151(83.4) | 27(14.9) * |
| 4 ℃ of refrigerations | 117 | 81(69.2) | 21(17.9) * |
Spilting of an egg rate=spilting of an egg number/cultivation embryo number; Blastaea rate=blastaea number/cultivation embryo number; Subscript different table differential different significantly (P＜0.05) in the same hurdle.
4 ℃ of refrigerations can make full use of more donorcells, and are particularly important when carrying out the preparation of transgene clone pig, enriched the stand-by mode of nuclear transplantation donorcells.
2, oocyte in vitro maturation
Get the ovary of gilt before puberty from the slaughterhouse, put into 30-35 ℃ physiological saline (adding penicillin 100,000 IU/ liters, Streptomycin sulphate 50mg/ liter), transport the laboratory in the 2h back.Ovarian follicle with 3-6mm on the 20ml syringe pump ovary of being furnished with No. 18 syringe needles.To extract liquid and be put in 37 ℃ of water-baths in the 50ml centrifuge tube,, put into the plastic culture dish of diameter 60mm more respectively then with 0.1%PVA-TL-HEPES washing 2 times.Under Stereo microscope, select ovarian cumulus parcel more than 3 layers, the fine and close and uniform cumulus cell of kytoplasm-ovocyte complex body (Cumulus-oocyte-complexes respectively with the mouth suction pipe, COCs) (Fig. 1), change in incubator at least (per 100 μ l drops are put 25 pieces) in maturation culture solution drips in the plastic culture dish of balance 4h for 3 times again over to oocyte in vitro maturation culture solution washing, do 4 drops in the plastic culture dish of each diameter 35mm, cover with embryo's level mineral oil.COCs cultivates earlier 20 ± 2h in oocyte in vitro maturation culture solution; Renew then in the droplet of bright oocyte in vitro maturation culture solution and continue to cultivate 20 ± 2h, experimental design is 2 * 2 experiment: (NCSU-23 or PZM-3 add 10% (volume ratio) pig follicle liquid (PFF) to above-mentioned 2 kinds of different oocyte in vitro maturation culture solution, 10ng/ml Urogastron (EGF), 10IU/ml human chorionic gonadotrophin (hCG) and 10IU/ml pregnant mare serum gonadotrop(h)in (PMSG) (PMSG)) and 2 kinds of different oxygen concentrations conditions, these 2 kinds of different oxygen concentrations conditions are respectively: contain 5% (volume ratio) CO
2Air (contain 20% (volume ratio) O approximately
2) or 7% (volume ratio) O
2, 5% (volume ratio) CO
2, 88% (volume ratio) N
2Gas mixture; Temperature is 39 ℃, and humidity is 100%.After maturation is cultivated termination, slough cumulus cell with taking off ovarian cumulus liquid.Transfer to the mature oocyte (Fig. 2) of discharging first polar body under stereoscope, statistical counting is analyzed the maturation in vitro rate.
Maturation culture solution and oxygen concn to the oocyte maturation rate to influence the result as shown in table 2, the result shows that oocyte maturation cultivates in the NCSU-23 substratum hyperoxia (20%O
2) and hypoxemia (7%O
2) maturation of oocyte nuclei there is not influence (84.8%vs.80.1%), and oocyte maturation is cultivated in the PZM-3 substratum, hyperoxia (20%O
2) descend the maturation of oocyte nuclei to be significantly higher than hypoxemia (7%O
2) (P＜0.05,75.4%vs.58.7%).
Different maturation culture solutions of table 2 and oxygen partial pressure influence oocyte maturation
*20%: contain 5%CO
2Air; 7%:5%CO
2, 7%O
2, 88%N
2.Subscript different table differential different significantly (P＜0.05) in the same hurdle.
3, maturation in vitro Activation of Oocyte and vitro culture
The above-mentioned preferably maturation in vitro ovocyte of form was transferred in fusion/activation liquid balance 20 seconds, after fusion/activation liquid washing 3 times, (electrode width is 500 μ m to put into the integration slot that is paved with fusion/activation liquid about 40 every batch, U.S. BTX), apply one 30 μ s with ECM2001 fusion instrument (BTX), the electric pulse of 1.2KV/cm is induced activation.The ovocyte that taking-up is handled well in NCSU-23+4mg/mL BSA+10 μ g/ml CB (cytochalasin B) liquid after the washing 3 times, changes that paraffin oil covers over to and in advance at CO
2Incubator (5%CO
2Air, temperature is 39 ℃, humidity is 100%) in cultivated 3-5 hour in 2 hours the NCSU-23+4mg/ml BSA+10 μ g/ml CB droplet of balance, take out again with NCSU-23+4mg/ml BSA give a baby a bath on the third day after its birth all over after transfer to paraffin oil and cover also in advance at CO
2(contain 5%CO in the incubator
2Air, temperature is 39 ℃, humidity is 100%) continue to cultivate at least 2 hours embryo medium of balance (NCSU-23+4mg/ml BSA or PZM-3+0.3% (0.3g/100ml) BSA) droplet.The drop size is 50 μ L, puts 20-25 ovum in each drop.Gas phase condition is for containing 5%CO
2Air in (hyperoxia) or 7%O
2, 5%CO
2, 88%N
2In the gas mixture of (hypoxemia), 39 ℃, 100% humidity is cultivated 168h observed and recorded blastaea rate.Embryo culture is 5%CO in NCSU-23
2Air and 7%O
2, 5%CO
2, 88%N
2Gas mixture cultivate 168 hours blastaea rates and be respectively 28.5 ± 7.5 and 19.7 ± 2.5,5%CO in PZM-3
2Air and 7%O
2, 5%CO
2, 88%N
2Gas mixture cultivate 168 hours blastaea rates and be respectively 18.7 ± 4.7 and 26.1 ± 4.6.The lonely female activation fetal development influence of maturation culture solution and oxygen partial pressure is as shown in table 3, be observations behind the 168h in the table 3, sophisticated ovocyte is to the blastaea rate of parthenogenetic embryo and blastomere number do not make significant difference (table 3) under the basic medium NCSU-23 of two kinds of nutrient solutions and PZM-3 hyperoxia and the hypoxemia.
Table 3 maturation culture solution and oxygen partial pressure are to the influence of the female activation fetal development of orphan
20%O
2: contain 5%CO
2Air; 7%O
2: 5%CO
2, 7%O
2, 88%N
2.
4, body-cell neucleus transplanting
The donorcells of 4 ℃ of preservations in the step 1 and the mature oocyte of discharge first polar body are changed in the micrurgy drop (Hepes-NCSU-23 (no calcium)+7.5 μ g/ml CB (cytochalasin B)) simultaneously, on the micrurgy instrument, use fixedly suction pipe (external diameter 100-120 μ m) sticking ovocyte then, with the blind suction method of the stoning/entry needle stoning of internal diameter 15-25 μ m, draw the kytoplasm that first polar body and adjacent 10-20% may contain oocyte nuclei.Select the circular slick donorcells of diameter 15-20 μ m, put into ovum week crack from the stoning otch.30 ovocytes of every batch operation, the cell that after the end donorcells-ooecium matter is constituted is transferred among the NCSU-23+4mg/mL BSA (bovine serum albumin) (reconstruct ovum), at 39 ℃, contains 5% (volume ratio) CO
2Air 100% humidity incubator in recover 0.5h.
5, the reconstruct ovum merges and activates
Transfer to balance 2min in fusion/activation liquid with recovering good reconstruct ovum in batches, fusion/activation liquid is by 0.25M N.F,USP MANNITOL, 0.1mM calcium chloride, 0.1mM magnesium chloride, 0.5mM HEPES and 0.01%PVA form, put into the integration slot (electrode width is 500 μ m) that is paved with fusion/activation liquid for 5 every batch, make donorcells-recipient oocyte film contact surface parallel with the solid glass pin with electrode, use ECM2001 fusion instrument (BTX again, USA) apply one 50 μ s, 2.0KV/cm electric pulse induce and merge and activate simultaneously, afterwards the reconstruct ovum is washed with NCSU-23+4mg/mL BSA, change in the embryo medium of mineral oil covering, at 39 ℃, contain 5%CO
2Air (contain 20% O approximately
2), cultivate after 0.5-1 hour under 100% humidity and take out decision fusion.
6, body-cell neucleus transplanting embryo vitro culture
(1) embryo culture stage oxygen partial pressure and substratum are to the influence of pig nuclear transfer embryo growth
To merge the activated embryo changes over to respectively under following 6 kinds of different conditions and cultivate: 1) cultivate in the NCSU-23+4mg/mLBSA embryo culture drop, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2Gas mixture; 2) cultivate in the PZM-3+3mg/mL BSA embryo culture drop, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2Gas mixture; 3) cultivate in the NCSU-23+4mg/mL BSA embryo culture drop, culture condition is 39 ℃, and 100% humidity contains 5%CO
2Air; 4) cultivate in the PZM-3+3mg/mL BSA embryo culture drop, culture condition is 39 ℃, and 100% humidity contains 5%CO
2Air; 5) will merge the activated embryo and change G1.3 (GIII)+1mg/mL BSA over to and cultivated 3 days, and change over to then and continue among G2.3 (GIII)+5mg/ml BSA to cultivate 3 days, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2Gas mixture; 6) will merge the activated embryo and change G1.3 (GIII)+1mg/mL BSA over to and cultivated 3 days, and change over to then and continue among the G2.3+5mg/mlBSA to cultivate 3 days, culture condition is 39 ℃, and 100% humidity contains 5%CO
2Air conditions.The drop of per 30 μ L is cultivated 8-10 piece of reconstructed embryo, and statistics spilting of an egg rate situation and blastaea form result and blastomere number when the 168h that cultivates, blastomere number Hoechst33342 dyeing counting (Fig. 5).Statistics is analyzed P＜0.05 significant difference with the t check of statistical analysis software SAS8.2.
The influence that embryo culture stage oxygen partial pressure and substratum are grown the pig nuclear transfer embryo is as shown in table 4, hypoxemia, hyperoxia concentration (7%O
2And 20%O
2) and NCSU-23, PZM-3, three kinds of substratum of GIII to the influence of fetal development, at hypoxia condition (7%O
2) under improved embryo's blastaea rate and cell count (the hypoxemia blastaea rate among the PZM-3 is lower than hyperoxia), three kinds of substratum differences are not remarkable.Wherein, substratum NCSU-23 is at 7%O
2Under the gas phase, blastaea rate and blastomere number improve significantly (P＜0.05,17.7 ± 2.5%vs8.2 ± 1.2%; 46.3 ± 9.6vs31.0 ± 2.1), and PZM-3 can only significantly improve the cell count (P＜0.05,49.4 ± 9.8vs36.6 ± 2.1) of blastaea under low oxygen, to blastaea rate do not make significant difference (P＞0.05,10.9 ± 4.5%vs.11.2 ± 3.6%).GIII (7%O
2) can significantly improve the cell count of blastaea, mean number can reach 64.3.GIII (20%O
2) the blastaea rate and the cell count of cultivating the embryo all significantly reduce, blastaea rate and cell count are respectively 5.6 ± 5.2% and 43.2 ± 7.6.
GIII is recently according to people's the uterine tube and the embryo medium of uterine luminal fluid development, one is cultivated the substratum that changes liquid in two stages is G1.3/G2.3, can adapt to the different fetal development stages to energy (glucose) and amino acid whose needs, it can improve the quality that multiple animal embryo is grown.Above-mentioned experimental result shows the pig embryo at recoverable about 10% the blastaea of GIII (substratum that is fit to hypoxemia), and has greatly improved the cell count of blastaea, and the raising of cell count may be because it be a more complete substratum.G1.3/G2.3 has the equilibrated nutritive ingredient, and O.5mM the concentration of glucose be respectively and 3.15mM, contains pyruvic acid, lactic acid and the amino acid of suitable concentration and VITAMIN etc.NCSU-23 then is a step substratum that contains higher concentration glucose (5.5mM), glutamine and taurine, and prescription does not contain pyruvic acid, lactic acid, VITAMIN and other amino acid.Culture effect analysis to these three kinds of substratum will help further improving substratum.
The influence that table 4 different partial and embryo culture medium are grown the pig nuclear transfer embryo
*20%; Contain 5%CO
2Air; 7%:5%CO
2, 7%O
2, 88%N
2,
A, b, cThe different significant differences (P＜0.05) of subscript in the same hurdle
(2) add the influence of EGF among the embryo culture medium NCSU-23 to clone's embryonic development
To merge the activated embryo and change cultivation in the NCSU-23+4mg/mL BSA embryo culture drop over to, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2The mixed gas condition be cultured to 2-4 cell stage (36h).Add 10ng/mL EGF at 2-4 cell stage (36h) at NCSU-23+4mg/mL BSA, a part is not added EGF and is organized in contrast, and culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2The mixed gas condition be cultured to 168h time statistics blastaea form result and blastomere number.The interpolation of EGF is as shown in table 5 to the influence of clone embryos growth in the substratum, the result shows with the control group that does not add EGF and compares, the treatment group of adding 10ng/ml EGF in the substratum has significantly improved the blastaea rate of formation (P＜0.05,25.8 ± 7.6%vs.8.3 ± 8.3%) of clone embryos.
Add the influence of EGF among the table 5 embryo culture medium NCSU-23 to clone's embryonic development
| EGF concentration (ng/ml) | Blastaea number/spilting of an egg number (%) | Cell count (mean ± S.E.) |
| 0 | 8.3±8.3 b | 43.0±3.3 |
| 10 | 25.8±7.6 * | 40.8±1.8 |
A, bSubscript different table differential different significantly (P＜0.05) in the same hurdle.
This experiment shows oxygen partial pressure is reduced to 5-10% from 20%, has improved embryo's potentiality of development greatly, and the embryo provides possibility for mass production.Cultivate early stage clone embryos under hypoxemia (7% oxygen) condition, more near internal milieu, blastaea ectogenesis rate can reach 10-30%.Conventional cultural method is under 20% oxygen partial pressure, and general blastaea ectogenesis rate is no more than 20%, and possible hyperoxia can produce more oxyradical and be harmful to embryo's growth.In substratum NCSU-23, add the blastaea rate of formation that 10ng/ml EGF has significantly improved clone embryos simultaneously.
The cultivation of embodiment 2, clone pig
1, body-cell neucleus transplanting embryo's acquisition
Adopt the tissue block inoculation method to set up porcine fetus fibroblasts system, conventional passage and freezing preservation to the Chinese experimental that is in the 33rd day Gestation period (breeding period be 0 day) with miniature pig (fragrant pig).Used substratum is the DMEM solution of above-mentioned preparation.2-6 time the porcine fetus fibroblasts of waiting to go down to posterity grows to 90% when converging, with 0.25% (0.25g/100ml) trypsinase tissue digestion liquid ordinary method digestion, centrifugal, add the resuspended cell precipitation of 200 μ LDMEM solution at last, after 4 ℃ of refrigerators are preserved 12h, be used as nuclear transplantation donorcells (4 ℃ of refrigerations) again.
Get the ovary of gilt before puberty from the slaughterhouse, cultivate according to the maturation of the method for step 2 among the embodiment 1 being carried out ovocyte.After maturation is cultivated termination, slough cumulus cell with taking off ovarian cumulus liquid.Under stereoscope, transfer to the mature oocyte (Fig. 2) of discharging first polar body.Mature oocyte is carried out body-cell neucleus transplanting according to the method for step 4 among the embodiment 1 obtain the cell of donorcells-ooecium matter formation (reconstruct ovum).
Method according to embodiment 1 step 5 merges the reconstruct ovum and activation, to merge the activated embryo changes in the embryo culture drop, method according to embodiment 1 step 6, in NCSU-23+4mg/mL BSA substratum, cultivate, the drop of per 30 μ L is cultivated 8-10 piece of reconstructed embryo, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2Cultivate 36h to 2-4 cell stage (Fig. 3).A part continues CO in the droplet of NCSU-23+4mg/mL BSA interpolation 10ng/mL EGF behind the 2-4 cell stage
2Support in the case and cultivate, culture condition is 39 ℃, 100% humidity, 7%O
2, 5%CO
2, 88%N
2The mixed gas condition, observe the situation of fetal development after cultivating 168h to blastaea, obtain body-cell neucleus transplanting blastaea (Fig. 4).
2, embryo transfer and pregnancy maintenance and detection
On the same day that makes up clone's embryo, select 3 multiparity binary sows (landrace * Large White, Large White * landrace) of spontaneous estrus: B792, B790 and P1652, the standard that begins to oestrus is the reflection of standing.Before embryo transfer next day, the conventional anesthesia of Thiopental Sodium, in operation Baoding of lying on one's side on the frame, an operative incision that is about 8cm is done by flank portion, expose ovary, uterine tube and uterus to the open air, (Agtech Inc.USA) enters about 5cm along fimbriae tubae with the embryo transfer pipe, and the body-cell neucleus transplanting body early embryo (cultivating 12-30h) that step 1 obtains is transplanted to ampulla of uterine tube-isthmus junction.B792 acceptor sow has been transplanted 150 pieces of clone embryos, has transplanted a small amount of embryo (20 pieces) after this friendship of B790 acceptor sow, and P1652 acceptor sow has been transplanted the clone embryos of moderate quatity (60 pieces).Treated pregnant sow intramuscular injection 1000IU PMSG (Tianjin Hua Fugao Newbiotics Inc) after the embryo transfer on the 10th day, the 13rd day injection 800IU hCG (the Ningbo second hormone factory).(whether Pie Medical Holand/Netherland) detects gestation to 30 days B ultrasounds, and the result shows B792 and B790 gestation after the embryo transfer.Then in the 50th day, 70 days and 90 days ultrasonic wave tracking detection fetation situations.The B792 pregnancy of wherein all transplanting clone embryos is mature, and in the rare alcohol-induced childbirth of the 115th day injection prostate gland, in the 116th day following 3 the black piglets of natural birth (table 6), the production efficiency of clone pig was 1.3% (3/230).A wherein survival (Fig. 6), very healthy always after the birth.
Grow in the body after the embryo transfer of table 6 clone embryo
| Acceptor sow (fate of oestrusing) | Clone's embryo number | Ultrasonic wave gestation detects | Produce the clone offspring |
| B792(1d) | 150 | Gestation | 3 black pigs (1 survival, 1 monster, 1 stillbirth) |
| B790(1d) | 20/this friendship | Gestation | 5 white pigs, but it is individual not have the clone |
| P1652(1d) | 60 | Nonpregnant | Do not have |
3, microsatellite DNA analysis
Whether using the microsatellite DNA analytical method, to detect the genotype of somatic cell clone pig consistent with donorcells, 2 clone pigs that the DNA sample source is produced in fragrant porcine fetus fibroblasts system (donorcells system), the B792 of step 1 and ear's tissue and parturient sow (landrace * Large White) the ear tissue of 1 stochastic sampling and sharp the organizing of piglet (landrace * Large White) tail of 2 stochastic samplings of acceptor sow B792.Choose 7 pairs of fluorescently-labeled micro-satellite primers (S0070, S0714, S072, SW1111, S742, S830 and S857) that are distributed in the coloured differently body, said gene group DNA is carried out pcr amplification by optimized reaction conditions.The PCR reaction system is in 20 μ l reaction systems, contains 10 * PCR damping fluid, 2 μ l, Taq enzyme 1.0U, 20ng/ μ L dna profiling 1 μ L, and every kind of each 1.0 μ L of primer 10 μ mol/L, ddH20 supplies 20 μ l.The PCR reaction conditions is, 94 ℃ of sex change 5min, and 94 ℃ of 30s, 55 ℃ of (or 60 ℃) 30s, 72 ℃ of 30s totally 35 circulations, then 72 ℃ are extended 7min, last 4 ℃ of insulations.Pcr amplification product carries out electrophoresis on the full-automatic sequenator of ABI377, with software Genescan3.1 electrophoresis result is carried out gene type assay.
Little satellite qualification result shows that 7 pairs of microsatellite DNA analyses (table 7) confirm that the genotype of 2 clone pigs and donorcells system is in full accord, and do not have any relevant with the parturient sow of acceptor sow, stochastic sampling, the piglet of stochastic sampling.The genetic material that clone pig is described is fully from donorcells.
7 microsatellite locus genotype of table 7 donorcells, replace-conceive sow and clone pig
| S0070 | S0714 | S072 | SW1111 | S742 | S830 | S857 | |
| Donorcells system | 274/290 | 124/128 | 114/116 | 168/174 | 199/199 | 185/187 | 143/151 |
| Acceptor sow B792 | 266/266 | 126/126 | 108/116 | 166/174 | 205/205 | 181/181 | 147/150 |
| Clone pig 1 | 274/290 | 124/128 | 114/116 | 168/174 | 199/199 | 185/187 | 143/151 |
| Clone pig 2 | 274/290 | 124/128 | 114/116 | 168/174 | 199/199 | 185/187 | 143/151 |
Claims (7)
1. method of producing somatic cell clone pig, be to be the nuclear transplantation donorcells with the porcine fetus fibroblasts, with at the NCSU-23 of no BSA or not have puberty father's former wife's porcine oocytes of cultivating the maturation in vitro that obtains in the PZM-3 maturation culture solution of BSA be the nuclear transplantation recipient cell, the nuclear transplantation donorcells is moved into non-nucleus egg mother cell be built into clone embryos, described clone embryos is 7% O at volumn concentration
2, 5% CO
2With 88% N
2The mixed gas gas phase condition under carry out vitro culture and obtain body early embryo, described body early embryo is implanted into treats pregnant sow gestation and produce somatic cell clone pig; The NCSU-23 of described no BSA or the PZM-3 maturation culture solution that does not have a BSA be NCSU-23 or PZM-3 nutrient solution to have added volume ratio be that 10% pig follicle liquid, concentration are the Urogastron of 10ng/ml, concentration is that human chorionic gonadotrophin and the concentration of 10IU/ml is the pregnant mare serum gonadotrop(h)in (PMSG) of 10IU/ml.
2. method according to claim 1 is characterized in that: described porcine fetus fibroblasts is handled through following method: existing digestion existing with or digestion after 2-8 ℃ of refrigeration 2-24 hour again.
3. method according to claim 1 is characterized in that: the used basic medium of described somatic cell clone embryo's vitro culture is NCSU-23, PZM-3 or GIII.
4. method according to claim 3 is characterized in that: the used basic medium of described somatic cell clone embryo's vitro culture is NCSU-23.
5. method according to claim 4 is characterized in that: the substratum of described somatic cell clone embryo's vitro culture is NCSU-23, PZM-3 or GIII in the somatic cell clone fetal development to the 2-4 cell stage; Behind the 2-4 of somatic cell clone fetal development cell stage and 2-4 cell stage NCSU-23, PZM-3 or the GIII that adds 1-10ng/ml EGF.
6. method according to claim 5 is characterized in that: the substratum that described somatic cell clone embryo's vitro culture is used is NCSU-23 in the somatic cell clone fetal development to the 2-4 cell stage; Behind the 2-4 of somatic cell clone fetal development cell stage and 2-4 cell stage the NCSU-23 that adds 1-10ng/ml EGF.
7. according to claim 5 or 6 described methods, it is characterized in that: the interpolation concentration of described EGF is 10ng/ml.
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| CN108060117B (en) * | 2017-12-01 | 2021-08-20 | 温氏食品集团股份有限公司 | Method for improving pig cloned embryo development efficiency |
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| CN1489899A (en) * | 2002-10-18 | 2004-04-21 | 中国人民解放军军事医学科学院生物工 | Rearing process for Shuangji cloned pig by gene engineering technology |
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