CN102766655A - Method for producing somatic cell cloned bovine blastocyst - Google Patents
Method for producing somatic cell cloned bovine blastocyst Download PDFInfo
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- CN102766655A CN102766655A CN2012102118174A CN201210211817A CN102766655A CN 102766655 A CN102766655 A CN 102766655A CN 2012102118174 A CN2012102118174 A CN 2012102118174A CN 201210211817 A CN201210211817 A CN 201210211817A CN 102766655 A CN102766655 A CN 102766655A
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Abstract
he invention discloses a method for producing somatic cell cloned bovine blastocysts. According to the method, to bovine ear limbal fibroblasts are used as nuclear transfer donorcells, and mature bovine oocytes cultivated in vitro are used as nuclear transfer receptor cells; the oocytes are denucleated by extrusion and removed with the zona pellucida; the oocytes and the donor cells are adhered in an arrangement way of oocytes-donor cells-oocytes and treated with electro-fusion to obtain reconstructed embryos; and the reconstructed embryos are activated and cultured in vitro to obtain the cloned blastocysts. The method can improve the production efficiency of the cloned bovine blastocysts.
Description
Technical field
The present invention relates to a kind of method of producing somatic cell clone ox blastaea.
Background technology
The mammalian somatic cell clone is meant somatic cell nuclear is moved in the stoning recipient cell (mature oocyte or zygote) that new reconstructed embryo is programmed again, divides, broken up and grow by the genetic information of donor.Comparatively the popular body-cell neucleus transplanting method is manual PCR cloning PCR and micrurgy at present.
The efficient of clone technology itself receives the influence of each link, comprises that the stoning of ovocyte, somatocyte inject ovocyte, enucleation oocyte and somatic fusion, the activation of reconstructed embryo, the vitro culture of reconstructed embryo.
The mature oocyte stoning is an important step in the nuclear transfer technology in clone's process, requires to go fully, reduces the damage of pair cell matter again as much as possible.Patterning method is adopted in manual PCR cloning PCR stoning, and its pair cell damage is big, and mortality ratio is higher; The microinjection rule is needled into operation in the ovocyte matter; The destructiveness of pair cell skeleton and film is stronger; And siphon away during stoning, comprise chromosomal tenuigenin and generally account for about 1/4th of ovocyte TV, cause losing in a large number of ovocyte matter.In addition, also have fluorescence guiding stoning method, blind suction method etc.Fluorescence guiding stoning method, reports (Critser etc., 1986) that irradiation time can not surpass 20 seconds under the fluorescence irradiation owing to use the fluorescence irradiation that ovocyte is had certain infringement, otherwise developmental potency descends or forfeiture.Blind suction method is meant the method (McGrath etc., 1983) with McGrath and Solter, promptly near the position of ovocyte discharging first polar body with nuclear substance in the cytolemma and the sucking-off together of parts of fine kytoplasm.Because except that mouse with the rabbit, the mature oocyte metaphase plate of other large animal can't be seen at microscopically, so stoning blindly.Because the interference of granulosa cell or first polar body are degenerated or ovum is aging, make chromosome position and first polar body depart from, so stoning of blind suction method and not exclusively success.
Fusion is another important step in the animal cloning technical process, and domestic method is electricity irritation.The principle that electricity merges utilizes high-voltage electric field on dotter haut and donor cell film, to hit some micropores simultaneously exactly, and dotter haut that is close together like this and donor cell film just merge through these micropores, supplies nuclear to introduce in the enucleated oocyte matter the most at last.Fusion rate is low in the microinjection, average out to 70-80%, and the failure of fusion causes embryo's the reduction with cloning efficiency of losing.
Somatic cell clone technique is all succeedd in the domestic animal of a plurality of kinds, and existing thousands of the somatic cell clone oxen in the whole world are born even to this day.However, the somatic cell clone efficient of ox is still very low, therefore, starts with from the key link of somatic cell clone, and the working method of developing a kind of high efficiency somatic cell clone ox blastaea is significant.
Summary of the invention
The technical problem that the present invention will solve is the method that a kind of high efficiency production somatic cell clone ox blastaea is provided.
The method of production somatic cell clone ox blastaea provided by the present invention may further comprise the steps:
(1) be the nuclear transplantation donorcells with the ears of an ox or cow edge inoblast;
(2) bovine oocyte is carried out maturation in vitro and cultivate, vitro culture is the ripe and ovocyte of discharging first polar body is as the nuclear transplantation recipient cell;
(3) step (2) gained ovocyte is pushed stoning and removes zona pellucida;
(4) earlier one piece of step (3) gained ovocyte and one piece of donorcells are sticked the formation complex body with the PHA method; Impose alternating-current; Again another piece step (3) gained ovocyte is attached on the above-mentioned complex body, it is put in order be ovocyte-donorcells-ovocyte; Impose direct current at last it is merged, make up the reconstruct embryo;
(5) after said reconstruct embryo activates, carry out vitro culture DCRP blastaea.
In the aforementioned production method, said the ears of an ox or cow edge fibroblastic age in generation is 5-15 generation, before nuclear transplantation, handles through serum starvation or handles without serum starvation.
Said bovine oocyte be by diameter be gather in the ox ovarian follicle of 2-8mm, by the ovocyte of the cumulus cell parcel of complete densification.
In the aforesaid method, the preparation method of the substratum that the said maturation in vitro of step (2) is cultivated is that the interpolation volume percent is 10% FBS in the TCM199 substratum, the FSH of 0.01IU/ml, the E2 of the LH of 0.01IU/ml and 1 μ g/ml.
The said extruding stoning of step (3) is to needle the zona pellucida at first polar body place at microscopically with top-notch kernel removing needle, and the kytoplasm of first polar body and below thereof is squeezed remove; The said zona pellucida that goes is to remove with pronase digestion.
The said electricity of step (4) merge with the said reconstruct embryo of step (5) activated pitch time be 1-3h, the preferred interval time is 2h.
The contriver has compared spilting of an egg rate and the blastaea rate that electric fusion and the activated timed interval are respectively the no zona pellucida clened cows embryo of 1h, 2h and 3h, and the result is as shown in table 1.The calculation formula of said spilting of an egg rate is: spilting of an egg rate=reconstructed embryo spilting of an egg number/fusion back reconstructed embryo number; The calculation formula of said blastaea rate is: blastaea rate=blastaea number/spilting of an egg embryo number.Down together.
Table 1, electricity merge and the influence of the reconstructed embryo activated timed interval to spilting of an egg rate and blastaea rate
| Treatment group | Handle the ovum number | Spilting of an egg number/rate (%) | Blastaea number/rate (%) |
| 1h | 136 | 119(88.05±8.06) | 37(31.45±3.07) |
| 2h | 153 | 117(78.79±25.67) | 53(43.29±13.59) |
| 3h | 177 | 135(76.62±11.67) | 40(32.95±8.82) |
Can get by table 1; The spilting of an egg rate of the treatment group of electricity fusion and reconstructed embryo activated timed interval 2h and 1h group, 3h organize no significant difference; But its blastaea rate will be higher than other two groups, prompting: electricity merges and the reconstructed embryo activated timed interval is that 2h can promote no zona pellucida reconstructed embryo to grow.
In the aforesaid method, the said reconstruct embryo's of step (5) activation is the activation of uniting of A23187 and 6-DMAP.
Said reconstruct embryo's vitro culture liquid is CR1aa, mCR1aa or SOFaaci, preferred mCR1aa nutrient solution.
The contriver has compared no zona pellucida clened cows embryo's in above-mentioned three kinds of reconstructed embryo vitro culture liquid spilting of an egg rate and blastaea rate, and the result is as shown in table 2.
Table 2, different nutrient solution do not have the influence that zona pellucida nuclear transplantation reconstructed embryo is grown to ox
| Nutrient solution | Handle the ovum number | Spilting of an egg number/rate (%) | Blastaea number/rate (%) |
| ?CR1aa | 102 | 61/(65.56±26.41) | 18/(29.74±1.37) |
| ?mCR1aa | 179 | 146/(83.17±4.76) | 41/(30.62±7.25) |
| ?SOFaaci | 106 | 42/(37.85±10.44) | 11/(26.19±4.12) |
Can get by table 2, the blastaea rate no significant difference of no zona pellucida reconstructed embryo in three kinds of nutrient solutions, but the spilting of an egg rate in the mCR1aa nutrient solution is higher than other two groups, and prompting: no zona pellucida clened cows embryo's developmental rate is higher in the mCR1aa nutrient solution.
The present invention will push pitting method first and combine to be applied to produce the somatic cell clone ox with no pellucid zone ovum parent cell technology, compare with traditional hand clone and microinjection, both improve fusion rate, improve spilting of an egg rate and blastaea rate again.At present, adopt this method successfully to obtain the somatic cell clone offspring of 1 He Sitan breeding oxen, follow-up also will have 8 somatic cell clone oxen to be born.
Description of drawings
Fig. 1 is the extruding stoning synoptic diagram of ovocyte of the present invention;
Fig. 2 pushes the chromosomal tenuigenin that comprises that stoning squeezes out for the present invention;
Fig. 3 is stoning of the present invention, no pellucid zone ovum parent cell and somatic fusion process, is followed successively by enucleation oocyte-donorcells complex body, the no zona pellucida reconstructed embryo that is merging from left to right, the no zona pellucida reconstructed embryo after merging;
Fig. 4 is the somatic cell clone ox embryo's of the present invention's preparation ectogenesis process; Last figure is followed successively by 1 cell stage, 2 cell stages, 4 cell stages from left to right, and figure below is followed successively by 8-16 cell stage, morula stage, blastaea from left to right;
Fig. 5 is the zona pellucida synoptic diagram of manual PCR cloning PCR 0.5% pronase digestion ovocyte;
Fig. 6 is the ovocyte after the manual PCR cloning PCR cutter cutting.
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
In following examples, the calculation formula of said fusion rate is: the logarithm of fusion rate=fusion back reconstructed embryo number/preceding half ovum of fusion; The calculation formula of said spilting of an egg rate, blastaea rate is said with preamble.
Embodiment 1 somatic cell clone legal system of the present invention is equipped with the clened cows blastaea
(1) preparation of donorcells
Get the ear edge inoblast of the He Sitan breeding oxen in 5-15 generation, when treating that cell grows to 70-80% and converges, change the DMEM nutrient solution that contains 0.5% foetal calf serum, continue to cultivate 3-5 days, subsequent use.
(2) collection of ovocyte and maturation in vitro are cultivated
Take the ox ovary from the slaughterhouse, put into the saline water about 30 ℃, in time transport the laboratory back.After ovary cleaned three times with saline water; Use the ovarian follicle of vacuum pump suction diameter as 2-8mm; Under stereoscopic microscope, select ovarian cumulus-ovocyte complex body (the cumulus-oocyte complexes that has complete densification; COCs), wash twice back with the no calcium magnesium PBS solution that contains 0.01%PVA earlier and move in the TCM199+10%FBS+0.01IU/mL FSH+0.01IU/mLLH+1 μ g/mL E2 maturation culture solution 50～60 pieces/hole.Place 38.5 ℃, the CO2gas incubator of 5%CO2, saturated humidity to cultivate 16-18h.Mature C OCs is removed granulosa cell with 0.1% Unidasa, select complete form then, kytoplasm evenly and ovocyte that first polar body occurs is arranged as the nuclear transplantation acceptor.
(3) the extruding stoning of ovocyte
The ovocyte that will have first polar body moves in the micrurgy liquid.Needle the zona pellucida at polar body place at 200 * microscopically with top-notch kernel removing needle, and the kytoplasm of first polar body and below thereof squeezed remove.Ovocyte after the stoning is put into the T10 droplet one by one, the kytoplasm bead in place to go part is detected whether contain nuclear and polar body through H33342 dyeing according to one-to-one relationship.Confirm the ovum of stoning put into T10 give a baby a bath on the third day after its birth all over after, with 0.5% pronase digestion removal zona pellucida.Then with T20 give a baby a bath on the third day after its birth all over after to put into incubator subsequent use, extruding stoning synoptic diagram is seen Fig. 1, the chromosomal tenuigenin that comprises that squeezes out is seen Fig. 2.
(4) reconstruct embryo's structure---no zona pellucida method
After earlier above-mentioned stoning, no pellucid zone ovum parent cell being handled 3～4s in 200 μ g/ml phytohemagglutinin (PHA) drops; Put it into again and contain man-to-man adherend cell in the somatic drop, select the median size volume, recover circular smooth in appearance as donorcells.Impose 3V alternating-current (AC); Make the orientation of somatocyte in the integration slot-ooecium matter complex body vertical with electrode automatically; Equally another stoning, no pellucid zone ovum parent cell are sticked on the complex body then; It is put in order be ovocyte-donorcells-ovocyte, and guarantee that its orientation is vertical with electrode.Impose 50V/mm at last, 9 μ s, the direct current of 1 pulse (DC) carry out electricity to it and merge, the statistics fusion rate.Fusion process is seen Fig. 3.
(5) reconstruct embryo's activation and vitro culture
After electricity merges 2h, reconstructed embryo moved in the mCR1aa nutrient solution that contains an amount of A23187 behind the effect 5min, cleans three times, move into again and continue in the mCR1aa nutrient solution that contains 2mM 6-DMAP to activate cultivation 6h, move at last and carry out vitro culture in the mCR1aa nutrient solution.The reconstructed embryo of no zona pellucida carries out vitro culture in little cave, the making in little cave is with reference to the Vajta reported method.Reconstructed embryo changes liquid and checks spilting of an egg rate situation at the about 48h of vitro culture; 7～8d inspection reconstructed embryo developmental state, statistics blastaea rate.
The somatic cell clone ox embryo's of method for preparing ectogenesis process is seen Fig. 4.
Embodiment 2 manual PCR cloning PCR preparation clone blastaeas
Wherein, the activation of the preparation of donorcells, the collection of ovocyte and maturation in vitro cultivation, reconstructed embryo and vitro culture step are with the corresponding steps among the embodiment 1, and ovocyte stoning and no pellucid zone ovum parent cell and somatic fusion process are following:
Manual stoning:
Ovocyte-cumulus cell complex body that maturation in vitro is cultivated about 18h is removed granulosa cell with 0.1% Unidasa; Place then and add 5% colcemid (Demecolcine; DC) in the ripe liquid; Select behind the effect 2h again and discharge ovocyte polar body, the tenuigenin homogeneous with 0.5% pronase digestion zona pellucida, under stereoscopic microscope, cut away bossed part with the micro-dissections cutter, it is subsequent use that seedless ovocyte is put into the T20 drop.See Fig. 5 and 6.
No pellucid zone ovum parent cell and somatic fusion:
After earlier half ovum being handled 3～4s in 200 μ g/ml phytohemagglutinin (PHA) drops, put it into again and contain man-to-man adherend cell in the somatic drop, select the median size volume, recover circular smooth in appearance as donorcells.Impose 3V alternating-current (AC); Make the orientation of somatocyte in the integration slot-ooecium matter complex body vertical with electrode automatically; Equally another ooecium matter is sticked on the complex body then; It is put in order be ovum-somatocyte-ovum, and guarantee that its orientation is vertical with electrode.Impose 50V/mm at last, 9 μ s, the direct current of 1 pulse (DC) carry out electricity to it and merge.Merge back 30min and detect the fusion situation, the statistics fusion rate.
The reconstructed embryo that success is merged activates and vitro culture, and its method steps is with embodiment 1, during statistics spilting of an egg rate and blastaea rate.
Embodiment 3 microinjections preparation clone blastaea
Wherein, the activation of the preparation of donorcells, the collection of ovocyte and maturation in vitro cultivation, reconstructed embryo and vitro culture step are with the corresponding steps among the embodiment 1, and the building process of ovocyte stoning, reconstructed embryo is following:
Micro-stoning:
The ovocyte that will have first polar body moves in the operation liquid H199+7.5ug/mL cytochalasin B droplet, under 20 power microscopes with the glass pipette (being with sharp) of 20 μ m diameters with first polar body with and the ovocyte of below in karyomit(e) absorb in the lump.Ovocyte after the operation moves in the droplet in corresponding another ware; Contain chromosomal kytoplasm with the Hoechst33342 5min that dyes, UV light is inspection stoning rate down, stoning completely ovocyte in T20 solution, give a baby a bath on the third day after its birth all over after; Put into 38.5 ℃, subsequent use in the 5%CO2 case.
The structure of reconstructed embryo:
(1) micro-notes nuclear
With enucleation oocyte with during donorcells immigration operation is dripped in right amount; Using an internal diameter to annotate nuclear pin absorption diameter as the sharp angle of hanging of 20-25 μ m is strong, circular, the slick donorcells of 15～20 μ m, refractivity; The ovum week that is injected into enucleation oocyte through the otch that stays during stoning on the zona pellucida is in the crack; Make the donorcells of injection be close to dotter haut, reconstructed embryo is put into embryo medium and is recovered 1～2h as far as possible.
(2) electricity of reconstructed embryo merges
Put into fusion liquid balance 2min with recovering good reconstructed embryo in batches, in integration slot, rotate ovocyte with dialling the ovum pin, make donorcells vertical with direction of an electric field with the ovocyte contact surface, imposing field intensity simultaneously is 2.2kVcm
-1, the burst length is 20 μ s, pulse number is that 1 time DC pulse merges (ECM-2001BTX), and behind the recovery 30min, the statistics fusion rate.
Select reconstructed embryo after the fusion and carry out next step and activate and handle and vitro culture, during statistics spilting of an egg rate and blastaea rate.
The foregoing description 1,2,3 clone's blastaea preparing methods' fusion rate, spilting of an egg rate and blastaea rate are as shown in table 3:
Table 3, different IPs implantation method are to the influence of transgene clone efficient
| The nuclear transplantation method | The reconstructed embryo number | Fusion rate | Spilting of an egg rate | The blastaea rate |
| The inventive method | 324 | 95.24%±1.58 | 90.56%±2.74 | 38.35%±4.13 |
| Manual clone | 153 | 90.33%±2.29 | 82.97%±2.49 | 32.36%±1.39 |
| Microinjection | 251 | 72.94%±7.92 | 89.10%±5.76 | 33.59%±2.25 |
Can be got by table 3, the fusion rate of the inventive method, spilting of an egg rate, blastaea rate all are higher than manual PCR cloning PCR and microinjection, show that the inventive method has higher bovine somatic cells cloning efficiency.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (9)
1. method of producing somatic cell clone ox blastaea may further comprise the steps:
(1) be the nuclear transplantation donorcells with the ears of an ox or cow edge inoblast;
(2) bovine oocyte is carried out maturation in vitro and cultivate, vitro culture is the ripe and ovocyte of discharging first polar body is as the nuclear transplantation recipient cell;
(3) step (2) gained ovocyte is pushed stoning and removes zona pellucida;
(4) earlier one piece of step (3) gained ovocyte and one piece of donorcells are sticked the formation complex body with the PHA method; Impose alternating-current; Again another piece step (3) gained ovocyte is attached on the above-mentioned complex body, it is put in order be ovocyte-donorcells-ovocyte; Impose direct current at last it is merged, make up the reconstruct embryo;
(5) after said reconstruct embryo activates, carry out vitro culture DCRP blastaea.
2. the method for claim 1 is characterized in that, said the ears of an ox or cow edge fibroblastic age in generation is 5-15 generation, before nuclear transplantation, handles through serum starvation or handles without serum starvation.
3. the method for claim 1 is characterized in that, said bovine oocyte be by diameter be gather in the ox ovarian follicle of 2-8mm, by the ovocyte of the cumulus cell parcel of complete densification.
4. the method for claim 1; It is characterized in that the preparation method of the substratum that the said maturation in vitro of step (2) is cultivated is that the interpolation volume percent is 10% FBS in the TCM199 substratum; 0.01IU/ml FSH, the E2 of the LH of 0.01IU/ml and 1 μ g/ml.
5. the method for claim 1 is characterized in that, the said extruding stoning of step (3) is to needle the zona pellucida at first polar body place at microscopically with top-notch kernel removing needle, and the kytoplasm of first polar body and below thereof is squeezed remove; The said zona pellucida that goes is to remove with pronase digestion.
6. the method for claim 1 is characterized in that, said electricity merge with said reconstruct embryo activated pitch time be 1-3h.
7. method according to claim 6 is characterized in that, said electricity merge with said reconstruct embryo activated pitch time be 2h.
8. method according to claim 1 is characterized in that, said reconstruct embryo's activation is the activation of uniting of A23187 and 6-DMAP.
9. the method for claim 1 is characterized in that, said reconstruct embryo's vitro culture liquid is CR1aa, mCR1aa or SOFaaci.
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| CN111690684A (en) * | 2020-04-30 | 2020-09-22 | 广州再生医学与健康广东省实验室 | Composition and application thereof |
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| CN113061572A (en) * | 2021-04-21 | 2021-07-02 | 广西壮族自治区畜牧研究所 | Micro-hole improvement method for culturing embryo without zona pellucida in manual cloning procedure |
| CN113174410A (en) * | 2021-04-21 | 2021-07-27 | 广西壮族自治区畜牧研究所 | Method for improving the means of donor-acceptor cell adhesion in manual cloning procedures |
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
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Application publication date: 20121107 |