CN105039305A - Improved method used for cloned embryo construction - Google Patents
Improved method used for cloned embryo construction Download PDFInfo
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- CN105039305A CN105039305A CN201510388055.9A CN201510388055A CN105039305A CN 105039305 A CN105039305 A CN 105039305A CN 201510388055 A CN201510388055 A CN 201510388055A CN 105039305 A CN105039305 A CN 105039305A
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Abstract
The invention belongs to the field of clone technology, and more specifically discloses an improved method used for cloned embryo construction. The improved method comprises following steps: S1, ovocytes are delivered into an operation droplet A, and blind absorption is adopted so as to realize denucleation; S2, cytoplasm obtained via denucleation is delivered into a dyed droplet, dyeing situations are observed directly under a fluoroscope, and the ovocytes processed via denucleation are reserved for standby application; S3, the ovocytes processed via denucleation are delivered into an operation droplet B for standing, and then are delivered into an operation droplet C containing a donor cells, one of the ovocyte is bound with one donor cell, and then is bound with another ovocyte so as to obtain a somatic cell-ovocyte-ovocyte combination; and S4, the somatic cell-ovocyte-ovocyte combination is allowed to stand in a fusion activation fluid, and then is delivered into a fusion slot for electrofusion. The improved method is simple and rapid in operation; denucleation is complete and high in efficiency; and cloning efficiency is high.
Description
Technical field
The present invention relates to clone technology field, be specifically related to improving one's methods of a kind of clone embryos structure.
Background technology
Animal cloning refers to that animal directly obtains the process with parent with identical inherited character offspring without amphigenetic mode, comprises parthenogenesis, blastomere separation and ientification, embryo separating and nuclear transplantation etc.At present, in fact the animal cloning technology be widely known by the people is exactly phalangeal cell nuclear transfer technology.Traditional nuclear transfer technology typically refers to by micrurgic method, in the mature oocyte nucleus of donorcells being injected stoning in advance or early stage zygote, form a new reconstructed embryo, reconstructed embryo genesis and development program after merging and activating is rearranged, through cell fission, differentiation develop into a new individuality in foster mothers.Comprise oocyte enucleation, note donorcells, merge donorcells and activate, vitro culture, the sequence of operations such as embryo transfer complex process, each operation steps can have influence on the final efficiency of nuclear transplantation.Since " Dolly " sheep birth in 1997, investigator improves each sport technique segment in nuclear transplantation process with regard to continuous.Handmade cloning (handmadecloning, HMC) technological improvement stoning operation steps, directly stoning is cut with special cutters under stereoscopic microscope, then close containing nucleolate half ovum and donorcells by two and impose electric pulse and make it merge, finally by single for reconstructed embryo cultivation.Whole process does not need micrurgy instrument, and Vajta etc. (Vajtaetal, 2001) are referred to as handmade cloning.Du Yutao (2011) research shows that, compared with traditional body-cell neucleus transplanting, handmade cloning technology can increase substantially the blastocyst rate of body-cell neucleus transplanting.
Got rid of by core in ovocyte matter, this process is referred to as stoning.The stoning of ovocyte is the key technique of somatic cell nuclear transfer technique, under removal as far as possible oligoplasmatic situation, ensure that stoning is clean simultaneously.If cytoplasm removal is too much, can make the protein of accumulation in ovocyte, mRNA runs off too much, affects the reprogrammed of donorcells.If stoning not exclusively can cause reconstructed embryo chromosomal aneuploidy, cause polyploid, the spilting of an egg is abnormal, impaired development, embryo's Deaths etc.The height of Enucleation efficiency is directly connected to the height of constructed clone embryos ectogenesis and acceptor transplant pregnancy rate and calving rate.
The pitting method of conventional cell nuclear transfer technology has and blindly sucks core method, dyeing stoning method and spindle-view method.But all there is certain shortcoming in often kind of method, people are seeking better method always.The extruding stoning method of such as (Liu Dong, 2004), pierces through an otch with glass needle zona pellucida near polar body, pushes ovocyte, makes first polar body overflow zona pellucida together with 1/4 ~ 1/3 kytoplasm near it.(Guo Jitong, 2002) carry out short-term culture with sucrose hypertonic solution to bovine oocyte, and the spindle body position of most of ovocyte protrudes from egg membrane surface, instruction stoning operation.These are all improve the stoning working method of ovocyte in trial.The handmade cloning technology (Vajta2007) grown up afterwards directly excises half ovum, to reach the clean object of stoning with cutter.
The blind core method (Fig. 2) that sucks is feature according to mature oocyte, remove about 1/3 kytoplasm near polar body, reach stoning object, operation is rapidly succinct, but the position of ovocyte first polar body can be made in After Enucleation to change, affect stoning effect, Enucleation efficiency is relatively low, generally 70%.Staining stoning utilizes DNA specific dye Hoechest33342 by after whole ovocyte dyeing, by the method for fluorescent microscope display karyomit(e) stoning, can remove a small amount of kytoplasm and ensure that 100% goes totally, but ultraviolet irradiation can damage embryonic itality (Tsunda, 1988).The spindle-view method developed afterwards, be utilize polarized light to reflect directly to obtain spindle body mirror image, do not need the spindle body just can observed in ovocyte that dyes, stoning rate can reach more than 95%, but expensive, is unfavorable for applying.Manual stoning is directly cut with cutter, the kytoplasm more (Fig. 1) relatively removed, and then 2 piece of half ovum fusion is made up the too much situation of removal kytoplasm.
Summary of the invention
Technical problem to be solved by this invention is, in order to overcome above-mentioned deficiency of the prior art, what provide a kind of clone embryos to build improves one's methods.
Above-mentioned technical problem to be solved by this invention is achieved by the following technical programs:
What a kind of clone embryos built improves one's methods, and comprises following steps:
S1. ovocyte is placed in operation drop A, utilizes blind suction method to complete stoning operation;
S2. nuclear for removal cytosolic fractions is placed in dyeing drop, directly at the coloring case of viewed under fluoroscopy kytoplasm, the ovocyte then by clean for the stoning of correspondence is for subsequent use;
S3. ovocyte clean for stoning is put into operation drop B to leave standstill, then move in the operation drop C be put into containing donorcells, first one piece of ovocyte and one piece of donorcells are bonded, and then be bonded together with another piece of ovocyte, it puts in order as somatocyte one ovum one ovum;
S4. somatocyte one ovum one ovum that bonded first is left standstill in fusion activation solution, then move on in integration slot and carry out electro' asion, i.e. DCRP embryo.
What clone embryos of the present invention built improves one's methods, and invention gropes to obtain through a large amount of experiments, and the method combines the advantage of several prior art, has abandoned its shortcoming; As absorbed the succinct advantage rapidly of blind suction method (Fig. 2) operation, overcome the shortcoming that its Enucleation efficiency is relatively low; Have employed the staining of improvement, absorb dyeing process stoning clean (stoning rate 100%) and the high advantage of efficiency, to turn avoid in traditional method ovocyte by UV-irradiation, the shortcoming of damage embryonic itality; Finally in conjunction with the handmade cloning technology of improvement, merge more kytoplasm, thus raise the efficiency.
Preferably, described ovocyte is porcine oocytes; Described donorcells is pig donorcells.
Preferably, the operation drop A described in S1. forms by without the H-NCSU-23 of calcium and 7.5 μ g/ml cytochalasin Bs (CB);
S2. the dyeing drop described in forms by without the H-NCSU-23 of calcium and the Hoechst33342 of 10 μ g/ml;
S3. the operation drop B described in is made up of the phytohemagglutinin (PHA) of H-NCSU-23 and 2mg/mL without calcium;
S3. the operation drop C described in is the H-NCSU-23 without calcium.
Preferably, the determination methods of the coloring case at viewed under fluoroscopy kytoplasm described in S2. is: if kytoplasm only has 1 point not shinny (Fig. 4), then prove that stoning is unclean; If kytoplasm has shinny (Fig. 5), then prove that corresponding oocyte enucleation is clean at 2.
Preferably, the time of repose described in S3. is 1 ~ 10s.
Preferably, the time of repose described in S4. is 1 ~ 5min.
Preferably, the fusion activation solution described in S4. is containing, for example lower component: 0.25mol/LMannitol, 0.1mmol/LCaCl22H2O, 0.1mmol/LMg-Cl26H2O, 0.5mmol/LHEPES, 0.01%PVA (w/v).
Preferably, the electro' asion described in S4. uses direct current to carry out, and parameter is: 80V/mm, 100 μ s, and pulse number is 2 ~ 3 times.
Preferably, in the integration slot described in S4., one end of the adjacent electrode of oocyte cytoplasm, somatocyte is positioned at the centre of integration slot.
Preferably, the stoning operation described in S1. and the complete operation in operation board of the dying operation described in S2.; Described operation board (Fig. 3) makes 4 rows, and often row is containing 5 drops, and wherein a row is containing operation drop, next-door neighbour
One row containing dyeing drop.
Beneficial effect: the invention provides a kind of brand-new clone embryos construction process, it has, and operation is succinct rapidly, stoning is clean and efficiency is high, cloning efficiency advantages of higher; In addition, the clone embryos obtained through the inventive method can significantly improve the blastocyst rate of vitro Development of Embryos.
Accompanying drawing explanation
Fig. 1 is manual stoning technology schematic diagram.
Fig. 2 is blind suction method stoning technology schematic diagram.
Fig. 3 is operation board figure.
Fig. 4 is sordid kytoplasm figure (the A white light of stoning under fluoroscope; B fluorescence).
Fig. 5 is kytoplasm figure (the A white light that under fluoroscope, stoning is clean; B fluorescence).
Fig. 6 is mirror blastomere counting diagram under fluorescence microscopy.
Embodiment
Explain the present invention further below in conjunction with specific embodiment, but embodiment does not limit in any form to the present invention.
Experimental study agents useful for same without specified otherwise all purchased from SIGMA company.Foetal calf serum (GIBCO), physiological saline (Ziguang Guhan Group Hengyang Pharmaceutical Co., Ltd); Cell cultures consumables associated therewith is Corning Products; Oocyte maturation and cell, embryo culture consumptive material are NUNC Products.Egg-cleaning liquid is DPBS+PVA liquid, and operation liquid is the H-NCSU-23 without calcium, and oocyte maturation nutrient solution is the TCM-199 liquid adding volume fraction 10% pig follicle liquid, and embryo medium is PZM-3.
(1) mature oocyte is obtained for subsequent use
Slaughterhouse gathers pig ovary, is placed in and transports laboratory back containing antibiotic 37 DEG C of physiological saline.With containing after antibiotic normal saline flushing 3-5 time, be the ovarian follicle of 2mm ~ 6mm with the 10mL syringe extraction diameter being furnished with 18G syringe needle.Under stereoscopic microscope with self-control pick up ovum pin pick kytoplasm complete and parcel more than 3 layers cumulus cells cumulus oocytes complesxes, 39 DEG C, 5%CO2, saturated humidity incubator in maturation culture 42h ~ 44h.Remove cumulus cell with 1mg/mL Unidasa, first polar body even with kytoplasm is discharged as maturity symbol picks out ripe ovocyte, is put in operation drop for subsequent use.
(2) donorcells is obtained for subsequent use
Gather pig ear sample, through containing dual anti-normal saline flushing, be saved in containing in dual anti-DMEM nutrient solution, take back laboratory with ice chest.Ear sample conveniently somatocyte is built system, method and is set up fibroblast, freezen protective.In 1-2 week before doing nuclear transplantation operation, donorcells recovery is cultivated.The Fibroblast cell-culture to 100% being passaged to 3-6 generation is converged, then contact inhibition is after 2 days, conventional digestion, centrifuge washing, finally with operation liquid, cell precipitation is resuspended, as nuclear donor.
(3) clone embryos builds
3.1 oocyte enucleation operations:
The operation of making 5*4 in operation board is dripped and dye droplet (Fig. 3), and first row is stoning operation drop (operation liquid+7.5 μ g/mlCB), and second row is dyeing drop (Hoechst33342 of operation liquid+10 μ g/ml).Put an ovocyte in each operation drop, utilize blind suction method to complete the stoning operation of ovocyte, the cytosolic fractions of removal is directly told at dyeing drop corresponding to second row.When whole stoning has operated, directly at the coloring case of viewed under fluoroscopy kytoplasm.Then the ovocyte by clean for the stoning of correspondence is for subsequent use.
The construction work of 3.2 clone embryos
Ovum clean for stoning is put into the operation drop 5s of the plant lectin (PHA) containing 2mg/mL, shift out the operation drop put into containing donorcells, being stirred by the thin glass needle of round end makes one piece of ovum first bond with one piece of donorcells, and then stick together with another piece of ovum, it puts in order as somatocyte one ovum one ovum.Somatocyte one ovum one ovum adhered to is first at fusion activation solution (0.25mol/LMannitol, 0.1mmol/LCaCl22H2O, 0.1mmol/LMg-Cl26H2O, 0.5mmol/LHEPES, 0.01%PVA (w/v)) in leave standstill 2min, then move on in integration slot and carry out electro' asion.Wherein one end of the adjacent electrode of oocyte cytoplasm, somatocyte is positioned at the centre of integration slot.Direct current merges activation parameter: 80V/mm, 100 μ s, 2DC, once can simultaneously fusion treatment 53 cell strings.
(4) vitro culture of embryo is reconstructed
The reconstructed embryo 5 μ g/mLCB+10 μ g/mLCHX assisted activation liquid process 4h after activating will be merged, reconstructed embryo is placed in the micro-cave of homemade WOW() four orifice plates, put into one piece of reconstructed embryo in each depression, 39 DEG C, 5%CO2, saturated humidity incubator in cultivate.
(5) ectogenesis of reconstructed embryo is observed
Embryo culture records spilting of an egg number and blastaea number to during 48h and 168h.Simultaneously to blastaea dyeing record blastomere number, because blastomere number is a prerequisite of reflection blastaea quality.Concrete grammar is: the blastaea taking out 6d, 10min is fixed wash 2 times in the DPBS containing 3.7% paraformaldehyde after, the blastaea fixed is transferred to lucifuge dyeing 5min in the DPBS containing the Hoechst33342 of 10 μ g/ml, good blastaea operation liquid is washed 3 times, transfer on slide glass, then gland slide gently.Then observe under ultraviolet excitation under Olympus fluorescent microscope and take pictures, cell counting (Fig. 6).Then carry out variance analysis to experimental data, the clone embryos construction process comparing improvement, on ectogenetic impact, comprises the difference of blastocyst rate and blastaea inner cell number.
embodiment 2
What we built with clone embryos of the present invention improves one's methods, and implements with reference to embodiment 1, compared with the handmade cloning of routine (with reference to embodiment 1, only step 3, clone embryos construction step is different), the results are shown in Table 1.Result shows, and the method for the invention, can significantly improve blastocyst rate, does not have significant difference between blastomere number, but has better growth trend.
Table 1. clone embryos construction process affects result table to vitro Development of Embryos
Note: the identical lowercase person of same column shoulder mark represents difference not significantly (P>0.05), the different lowercase person of same column shoulder mark represents significant difference (P<0.05).
Claims (10)
1. what a clone embryos built improves one's methods, and it is characterized in that, comprises following steps:
S1. ovocyte is placed in operation drop A, utilizes blind suction method to complete stoning operation;
S2. nuclear for removal cytosolic fractions is placed in dyeing drop, directly at the coloring case of viewed under fluoroscopy kytoplasm, the ovocyte then by clean for the stoning of correspondence is for subsequent use;
S3. ovocyte clean for stoning is put into operation drop B to leave standstill, then move in the operation drop C be put into containing donorcells, first one piece of ovocyte and one piece of donorcells are bonded, and then be bonded together with another piece of ovocyte, it puts in order as somatocyte one ovum one ovum;
S4. somatocyte one ovum one ovum that bonded first is left standstill in fusion activation solution, then move on in integration slot and carry out electro' asion, i.e. DCRP embryo.
2. according to claim 1ly improve one's methods, it is characterized in that, described ovocyte is porcine oocytes; Described donorcells is pig donorcells.
3. according to claim 1ly to improve one's methods, it is characterized in that, the operation drop A described in S1. forms by without the H-NCSU-23 of calcium and 7.5 μ g/ml cytochalasin Bs;
S2. the dyeing drop described in forms by without the H-NCSU-23 of calcium and the Hoechst33342 of 10 μ g/ml;
S3. the operation drop B described in is made up of the phytohemagglutinin of H-NCSU-23 and 2mg/mL without calcium;
S3. the operation drop C described in is the H-NCSU-23 without calcium.
4. according to claim 1ly improve one's methods, it is characterized in that, the determination methods of the coloring case at viewed under fluoroscopy kytoplasm described in S2. is: if kytoplasm only has 1 point not shinny, then prove that stoning is unclean; If kytoplasm have 2 shinny, then prove that corresponding oocyte enucleation is clean.
5. according to claim 1ly improve one's methods, it is characterized in that, the time of repose described in S3. is 1 ~ 10s.
6. according to claim 1ly improve one's methods, it is characterized in that, the time of repose described in S4. is 1 ~ 5min.
7. according to claim 1ly improve one's methods, it is characterized in that, the fusion activation solution described in S4. is containing, for example lower component: 0.25mol/LMannitol, 0.1mmol/LCaCl22H2O, 0.1mmol/LMg-Cl26H2O, 0.5mmol/LHEPES, 0.01%PVA (w/v).
8. according to claim 1ly to improve one's methods, it is characterized in that, the electro' asion described in S4. uses direct current to carry out, and parameter is: 80V/mm, 100 μ s, and pulse number is 2 ~ 3 times.
9. according to claim 1ly improve one's methods, it is characterized in that, in the integration slot described in S4., one end of the adjacent electrode of oocyte cytoplasm, somatocyte is positioned at the centre of integration slot.
10. according to claim 1ly to improve one's methods, it is characterized in that, the stoning operation described in S1. and the complete operation in operation board of the dying operation described in S2.; Described operation board makes 4 rows, and often row is containing 5 drops, and wherein a row is containing operation drop, and a row of next-door neighbour is containing dyeing drop.
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| CN107858376A (en) * | 2017-11-10 | 2018-03-30 | 南开大学 | A kind of measuring method of stoning amount |
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