CN106544368A - A kind of mouse embryo cell and preparation method thereof and No operation transfer method - Google Patents
A kind of mouse embryo cell and preparation method thereof and No operation transfer method Download PDFInfo
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Abstract
A kind of mouse embryo cell and preparation method thereof and No operation transfer method, belong to induced multi-potent embryonic stem cell field.The preparation method of the mice embryonic that the present invention is provided is by preparing, incubation culture donorcellses, the step such as cell fusion and embryonic cell culture and pick, can be by simple operational approach, quickly induce and prepare the embryonic cell of mice, the embryonic cell has preferable totipotency and differentiation capability, can meet the demand of various heredity, development experiment etc..
Description
Technical field
The present invention relates to induced multi-potent embryonic stem cell field, and more particularly to a kind of mouse embryo cell and its preparation side
Method and No operation transfer method.
Background technology
Mice is a kind of conventional laboratory animal in bioscience research.In field of pharmacology, generally make of mice embryonic
The metabolism research of related drugs, or pathogenetic research of the disease of correlation.
However, the supply of the embryo of mice is but usually insufficient, although the reproductive performance of mice is stronger, but if from bosom
Pregnant mice takes embryo；Both the problem having had in terms of some ethics, generally also can cause physiology or psychological to experimenter
Harmful effect.
The content of the invention
It is an object of the invention to provide a kind of preparation method of mice embryonic, the preparation method is simple to operate, condition temperature
With；The embryonic cell for preparing mice can quickly and easily be induced.
The second object of the present invention is to provide a kind of mouse embryo cell, and the mouse embryo cell possesses the all-round of development
Property, of many uses, cost is relatively low.
The third object of the present invention is the No operation transfer method for providing a kind of mice embryonic, and the method can be by simple
Operation, mouse embryo cell is transplanted to into the intrauterine of female mice, the totipotency of embryonic cell can be further verified.
The present invention solves its technical problem and employs the following technical solutions to realize.
A kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice；
Donorcellses incubation step：Co-cultured with epidermis cell and ovum with reprogramming the factor respectively, at -8 to -5 DEG C
Under conditions of be incubated 12-16min；
Cell fusion step：Oocyte enucleation is obtained into enucleated oocyte, the nucleus injection enucleation of epidermis cell is taken out
Ovum, and processed with fusion liquid, obtain embryo's source cell；
Embryonic cell incubation step：Embryo's source cell is cultivated in the culture bottle containing culture medium；
Embryonic cell screens step：In superclean bench, stereomicroscope screening inspection is used, find suitable embryo thin
Born of the same parents, embryonic cell is cleaned with embryo's mixed liquor.
A kind of mouse embryo cell, is obtained by the preparation method of above-mentioned mice embryonic.
Above-mentioned mouse embryo cell is developed to blastula stage by a kind of No operation transfer method of mouse embryo cell, will
The mouse cell of blastula stage loads grafts, and grafts are put in embryo transfer gun, cleans and dry recipient female mice
After pudendum, transplanting rifle is sent into into female mice intrauterine, mouse embryo cell is sent into recipient female little by insertion transplanting rifle inner core
Mus intrauterine.
Compared with prior art, the invention has the beneficial effects as follows：The preparation method of the mice embryonic that the present invention is provided passes through
Prepare, donorcellses are cultivated in incubation, the step such as cell fusion and embryonic cell culture and pick, the operational approach are simple,
Condition is simple；The embryonic cell prepared by the method, with preferable totipotency and differentiation capability；Blastula stage will be developed to
The No operation transfer method of mouse embryo cell can further verify the totipotency and differentiation capability of mouse embryo cell, widen little
The application of Mus embryonic cell.
Specific embodiment
To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional product that can pass through that commercially available purchase is obtained
Product.
Below a kind of mouse embryo cell of the embodiment of the present invention and preparation method thereof and No operation transfer method are made into
The explanation of one step.
A kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice；
Donorcellses incubation step：Co-cultured with epidermis cell and ovum with reprogramming the factor respectively, at -8 to -5 DEG C
Under conditions of be incubated 12-16min；
Cell fusion step：Oocyte enucleation is obtained into enucleated oocyte, the nucleus injection enucleation of epidermis cell is taken out
Ovum, and processed with fusion liquid, obtain embryo's source cell；
Embryonic cell incubation step：Embryo's source cell is cultivated in culture bottle, culture bottle is put in incubator and is cultivated.
Embryonic cell screens step：In superclean bench, stereomicroscope screening inspection is used, find suitable embryo thin
Born of the same parents, embryonic cell is cleaned with embryo's mixed liquor.
In donorcellses step is prepared, take that differentiation is normal and the epidermis cell of mice in vigorous period is divided,
Defective cell can be avoided to greatest extent to be applied to experiment；And sexually matured mice ovum is selected, the ovum energy
Adapt to the demand of the matter and energy at fetal development initial stage.
Further, in the preferred embodiment, in donorcellses incubation step, the reprogramming factor includes
At least one in Oct4, K1f4, c-Myc, Sox2, Lin28 or Nanog.
The nucleus process of rearranging includes the change of gene expression pattern and the change of epigenetic labelling.In somatic cell gram
In grand, oocyte must be wiped the differentiation in donorcellses core first and remember and set up the all-round state of similar embryo, this
Process needs epigenetic to rearrange (epigenetic reprogramming) to wipe epigenetic original in somatic cell nuclear
Pattern simultaneously sets up the epigenetic characteristic and gene expression pattern of embryo in clone embryos.Current viewpoint thinks, apparent something lost
It is to cause clone embryos development failure, the principal element that fetal development is less efficient and phenotype is abnormal to pass imperfection of rearranging.Therefore,
The epigenetic reprogramming of preimplantation embryo phase for correct development it is critical that, it can adjust body early embryo gene
Expression, cell division further determine cell fate.The reprogramming factor plays an important role during the epigenetic is rearranged,
Reprogramming factor Oct4, Sox2, c-Myc, Klf4, Lin28 and Nanog.Oct4, Sox2, c-Myc and Klf4 gene just can be into
Fibrocyte is rearranged becomes embryonic stem cell-like cell.Therefore, reprogramming the factor is used for somatic cell clone skill by researcher at present
In art, improve clone embryos reprogramming ability and then improve clone embryos development efficiency.
Further, in the preferred embodiment, in donorcellses incubation step, the reprogramming factor for using
Concentration be 85ng/ μ L-90ng/ μ L.
Further, in the preferred embodiment, in donorcellses incubation step, reprogram the concentration of the factor
For 87ng/ μ L.
By using the reprogramming factor that concentration is 85ng/ μ L-90ng/ μ L, and the preferably concentration of the reprogramming factor
For 87ng/ μ L, differentiated cell can be preferably stimulated to recover differentiation capability.
Further, in the preferred embodiment, in cell fusion step, fusion liquid includes based on per liter：
The Mannitol of BSA, 51.0-54.7g of 0.1-0.12g, the calcium acetate of 16-24mg, the potassium pyrophosphate of 33-99mg and 119.2-
The Hepes (4- hydroxyethyl piperazine ethanesulfonic acid) of 178.7mg.
In cell fusion liquid, cell can preferably keep its normal form, and the activity of cell also can be preferable
Keep.
Further, in the preferred embodiment, in embryonic cell fusion steps, the osmotic pressure for merging liquid is
245-252mOsm。
The infiltration for adjusting fusion liquid is depressed into the 245-252mOsm (millis of non-electrolyte or electrolyte contained by 1 liter of water of expression
Molal quantity), keep a cell under an isotonic state；Stablizing for the osmotic pressure of holding fusion liquid, is conducive to maintaining cell
The electrolyte balance of form and Cell sap, maintains the vigor of cell；Meanwhile, liquid is merged by 0.22 μm of membrane filtration and not only may be used
To remove impurity, moreover it is possible to degerming, it is to avoid cell is contaminated.
Further, embryonic cell screening step in, embryo's mixed liquor based on per liter, containing following component：The chlorination of 3-5g
Sodium, the potassium chloride of 0.1-0.3g, the potassium pyrophosphate of 1-3g, the disodium hydrogen phosphate of 1-1.5g, 0.2g potassium dihydrogen phosphates, 0.05-
The calcium chloride of 0.1g, the magnesium chloride of 0.1g.
Substantial amounts of electrolyte is added, stablizing for cell can be maintained, maintain the vigor of cell.
Further, in embryonic cell screening step, embryo's mixed liquor also contains based on per liter：The Sanguis Bovis seu Bubali of 3-6ml is pure
The calf serum of protein solution or 10-100ml；The penicillin of ten thousand units of 60-85, the kanamycin of ten thousand units of 60-85.
Add bovine serum albumin or calf serum to be cells with nutrient, make cell be maintained normal new old generation
Thank；And add penicillin or kanamycin effectively suppress the growth of antibacterial, to the ring of one safety and stability of cell growth
Border.
A kind of mouse embryo cell, is obtained by above-mentioned preparation method.
Above-mentioned mouse embryo cell is developed to blastula stage by a kind of No operation transfer method of mouse embryo cell, will
Mouse embryo cell in blastula stage loads grafts, and grafts are put in embryo transfer gun, and embryo transfer gun is sent
Enter the intrauterine of recipient female mice, mouse embryo cell is sent into the intrauterine of recipient female mice by insertion transplanting rifle inner core.
With reference to embodiments the feature and performance of the present invention are described in further detail.
Embodiment 1
The present embodiment provides a kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice.
The epidermis cell and ovum of the sexual maturity female mice of acquirement are added in cell culture medium, in temperature be 37
℃、CO2Concentration be 5% saturated humidity incubator in cultivate.
Donorcellses incubation step：The epidermis cell and ovum of culture are diluted to into 1 × 106/ mL, with concentration be
Reprogramming factor Oct4 of 85ng/ μ L is co-cultured with epidermis cell and ovum respectively, is incubated 12min at -5 DEG C.
Cell fusion step：Oocyte is suctioned out into the nucleus of ovum with injection needle, enucleated oocyte is obtained；
Same operation, takes out the nucleus of epidermis cell, and by the nucleus of epidermis cell injection enucleated oocyte, and with merging liquid
Process, obtain embryo's source cell.Fusion liquid based on per liter, including：The Mannitol of BSA, 51.0g of 0.1g, the calcium acetate of 16mg,
The Hepes of the potassium pyrophosphate and 119.2mg of 33mg；The osmotic pressure of fusion liquid is 245mOsm, and with 0.22 μm of membrane filtration.
Embryonic cell incubation step：Embryo's source cell is cultivated in culture bottle, culture bottle 37 DEG C is put into into, CO2Concentration
To cultivate in the incubator of 5% saturated humidity.
Embryonic cell screens step：In superclean bench, first embryonic cell is cleaned with embryo's mixed liquor multiple；Embryo mixes
Close liquid and contain following component：The Sodium Chloride of 3g, the potassium chloride of 0.1g, 1g potassium pyrophosphates, the disodium hydrogen phosphate of 1g, the di(2-ethylhexyl)phosphate of 0.2g
Hydrogen potassium, the calcium chloride of 0.05g, the magnesium chloride of 0.1g.Other embryo's mixed liquor also bovine serum albumin solution containing 3ml；60
The penicillin of ten thousand units, the kanamycin of 600,000 units.
Stereomicroscope screening inspection is used again, from right to left, can walk one " S " using from left to right followed by the right
The route of shape carries out inspection screening；Or from top to bottom, followed by following, from the bottom up, the route for walking " W " shape is examined
Look into screening；Suitable embryonic cell is found, embryonic cell is cleaned with embryo's mixed liquor.
Embodiment 2
The present embodiment provides a kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice.
The epidermis cell and ovum of the sexual maturity female mice of acquirement are added in cell culture medium, in temperature be 37
DEG C, CO2Concentration be 5% saturated humidity incubator in cultivate.
Donorcellses incubation step：The epidermis cell and ovum of culture are diluted to into 1 × 106/ mL, with concentration be
Reprogramming factor Oct4, Sox2 of 86ng/ μ L, c-Myc and Klf4.The reprogramming factor is trained altogether with epidermis cell and ovum respectively
Support, 13min is incubated at -6 DEG C.
Cell fusion step：Oocyte is suctioned out into the nucleus of ovum with injection needle, enucleated oocyte is obtained；
Same operation, takes out the nucleus of epidermis cell, and by the nucleus of epidermis cell injection enucleated oocyte, and with merging liquid
Process, obtain embryo's source cell.Fusion liquid includes：The Mannitol of BSA, 52g of 0.11g, the calcium acetate of 18mg, the burnt phosphorus of 55mg
The Hepes of sour potassium and 130mg；Fusion liquid osmotic pressure is 248mOsm, and with 0.22 μm of membrane filtration.
Embryonic cell incubation step：Embryo's source cell is cultivated in culture bottle, culture bottle is put in incubator and is cultivated.
Embryonic cell screens step：In superclean bench, first embryonic cell is cleaned with embryo's mixed liquor multiple；Embryo mixes
Liquid is closed in terms of per liter, containing following component：The Sodium Chloride of 4g, the potassium chloride of 0.2g, the potassium pyrophosphate of 2g, the phosphoric acid hydrogen two of 1.2g
Sodium, the potassium dihydrogen phosphate of 0.2g, the calcium chloride of 0.06g, the magnesium chloride of 0.1g.Embryo's mixed liquor also Sanguis Bovis seu Bubali containing 6ml is pure
Protein solution；The penicillin of 750000 units, the kanamycin of 850,000 units.
Stereomicroscope screening inspection is used again, from right to left, can walk one " S " using from left to right followed by the right
The route of shape carries out inspection screening；Or from top to bottom, followed by following, from the bottom up, the route for walking " W " shape is examined
Look into screening；Suitable embryonic cell is found, embryonic cell is cleaned with embryo's mixed liquor.
Embodiment 3
The present embodiment provides a kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice.
The epidermis cell and ovum of the sexual maturity female mice of acquirement are added in cell culture medium, in temperature be 37
DEG C, CO2Concentration be 5% saturated humidity incubator in cultivate.
Donorcellses incubation step：The epidermis cell and ovum of culture are diluted to into 1 × 106/ mL, with concentration be
Reprogramming factor Oct4, K1f4 of 87ng/ μ L, c-Myc, Sox2, Lin28 and Nanog.Reprogramming the factor respectively with epidermis cell
Co-culture with ovum, 14min is incubated at -7 DEG C.
Cell fusion step：Oocyte is suctioned out into the nucleus of ovum with injection needle, enucleated oocyte is obtained；
Same operation, takes out the nucleus of epidermis cell, and by the nucleus of epidermis cell injection enucleated oocyte, and with merging liquid
Process, obtain embryo's source cell.Fusion liquid includes：The Mannitol of BSA, 53.5g of 0.12g, the calcium acetate of 20g, Jiao of 77mg
The Hepes of potassium phosphate and 154mg；Fusion liquid osmotic pressure position 250mOsm, and with 0.22 μm of membrane filtration.
Embryonic cell incubation step：Embryo's source cell is cultivated in culture bottle, culture bottle is put in incubator and is cultivated.
Embryonic cell screens step：In superclean bench, first embryonic cell is cleaned with embryo's mixed liquor multiple；Embryo mixes
Liquid is closed in terms of per liter, containing following component：The Sodium Chloride of 5g, the potassium chloride of 0.3g, the potassium pyrophosphate of 2.5g, the phosphoric acid hydrogen two of 1.4g
Sodium, the potassium dihydrogen phosphate of 0.2g, the calcium chloride of 0.08g, the magnesium chloride of 0.1g.Embryo's mixed liquor also calf blood containing 10ml
Clearly；The penicillin of 850000 units, the kanamycin of 700,000 units.
Stereomicroscope screening inspection is used again, from right to left, can walk one " S " using from left to right followed by the right
The route of shape carries out inspection screening；Or from top to bottom, followed by following, from the bottom up, the route for walking " W " shape is examined
Look into screening；Suitable embryonic cell is found, embryonic cell is cleaned with embryo's mixed liquor.
Embodiment 4
The present embodiment provides a kind of preparation method of mice embryonic, comprises the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice.
The epidermis cell and ovum of the sexual maturity female mice of acquirement are added in cell culture medium, in temperature be 37
DEG C, CO2Concentration be 5% saturated humidity incubator in cultivate.
Donorcellses incubation step：The epidermis cell and ovum of culture are diluted to into 1 × 106/ mL, with concentration be
Reprogramming factor c-Myc and Sox2 of 90ng/ μ L.The reprogramming factor is co-cultured with epidermis cell and ovum respectively, at -8 DEG C
Incubation 16min.
Cell fusion step：Oocyte is suctioned out into the nucleus of ovum with injection needle, enucleated oocyte is obtained；
Same operation, takes out the nucleus of epidermis cell, and by the nucleus of epidermis cell injection enucleated oocyte, and with merging liquid
Process, obtain embryo's source cell.Fusion liquid in terms of per liter, including：The Mannitol of BSA, 54.7g of 0.12g, the acetic acid of 24mg
The Hepes of calcium, the potassium pyrophosphate of 99mg and 178.7mg；Fusion liquid osmotic pressure is 252mOsm, and with 0.22 μm of membrane filtration.
Embryonic cell incubation step：Embryo's source cell is cultivated in culture bottle, culture bottle is put in incubator and is cultivated.
Embryonic cell screens step：In superclean bench, embryonic cell 15 times is first cleaned with embryo's mixed liquor；Embryo mixes
Liquid is closed in terms of per liter, containing following component：The Sodium Chloride of 5g, the potassium chloride of 0.3g, the potassium pyrophosphate of 3g, the phosphoric acid hydrogen two of 1.5g
Sodium, the potassium dihydrogen phosphate of 0.2g, the calcium chloride of 0.1g, the magnesium chloride of 0.1g.Embryo's mixed liquor also calf blood containing 100ml
Clearly；The penicillin of 850000 units, the kanamycin of 600,000 units.
Stereomicroscope screening inspection is used again, from right to left, can walk one " S " using from left to right followed by the right
The route of shape carries out inspection screening；Or from top to bottom, followed by following, from the bottom up, the route for walking " W " shape is examined
Look into screening；Suitable embryonic cell is found, embryonic cell is cleaned with embryo's mixed liquor.
Embodiment 5
A kind of mouse embryo cell is present embodiments provided, the method system that the mouse embryo cell is provided by embodiment 4
It is standby and obtain.
Embodiment 6
The present embodiment provides a kind of mouse embryo cell No operation transfer method, and the method transfer is that embodiment 5 is provided
Mouse embryo cell.
First：By Recipient mice artificial Baoding, Recipient mice vaginal orifice portion is cleaned, by the reproductive tract of Recipient mice to receptor
The uterus of mice is inserted into embryo transfer gun.
Secondly：The embryonic cell of mouse embryo cell culture to blastula stage, culture fluid and blastula stage is made into embryonic cell
Suspension, proceeds in grafts.
Secondly：Grafts are inserted in embryo transfer gun, the embryonic cell of mice is sent into receptor by insertion transplanting rifle inner core
The intrauterine of female mice.
In sum, a kind of mouse embryo cell of the embodiment of the present invention and preparation method thereof and No operation transfer method,
By the preparation method of the mouse embryo cell, can quickly induce and prepare mice by simple operational approach
Embryonic cell, the embryonic cell has preferable totipotency and differentiation capability, can meet the need of various heredity, development experiment etc.
Ask；The embryonic cell of obtained mice is developed to into blastula stage, the intrauterine of mice is then proceeded to, the embryo of mice is further verified
The totipotency of fetus cells.
Embodiments described above is a part of embodiment of the invention, rather than the embodiment of whole.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected enforcement of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
1. a kind of preparation method of mice embryonic, it is characterised in that comprise the following steps：
Prepare donorcellses step：Take the epidermis cell and ovum of sexually matured female mice；
Donorcellses incubation step：Co-cultured with the epidermis cell and the ovum with reprogramming the factor respectively, -8 to -
12-16min is incubated under conditions of 5 DEG C；
Cell fusion step：The oocyte enucleation is obtained into enucleated oocyte, the nucleus injection of the epidermis cell is taken out
The enucleated oocyte, and processed with fusion liquid, obtain embryo's source cell；
Embryonic cell incubation step：Embryo's source cell is cultivated in the culture bottle containing culture medium；
Embryonic cell screens step：In superclean bench, stereomicroscope screening inspection is used, finds suitable embryonic cell,
The embryonic cell is cleaned with embryo's mixed liquor.
2. the preparation method of mice embryonic according to claim 1, it is characterised in that the donorcellses incubation step
In, the reprogramming factor includes at least one in Oct4, K1f4, c-Myc, Sox2, Lin28 or Nanog.
3. the preparation method of the mice embryonic according to any one of claim 1-2, it is characterised in that the donorcellses are incubated
Educate in step, the concentration of the reprogramming factor is 85ng/ μ L-90ng/ μ L.
4. the preparation method of mice embryonic according to claim 3, it is characterised in that the donorcellses incubation step
In, the concentration of the reprogramming factor is 87ng/ μ L.
5. the preparation method of mice embryonic according to claim 1, it is characterised in that in the cell fusion step, institute
Stating fusion liquid includes based on per liter：The Mannitol of BSA, 51.0-54.7g of 0.1-0.12g, the calcium acetate of 16-24mg, 33-
The Hepes of the potassium pyrophosphate and 119.2-178.7mg of 99mg.
6. the preparation method of mice embryonic according to claim 5, it is characterised in that the osmotic pressure position of the fusion liquid
245-252mOsm。
7. the preparation method of mice embryonic according to claim 1, it is characterised in that the embryonic cell screens step
In, embryo's mixed liquor includes based on per liter：The Sodium Chloride of 3-5g, the potassium chloride of 0.1-0.3g, the potassium pyrophosphate of 1-3g, 1-
The disodium hydrogen phosphate of 1.5g, 0.2g potassium dihydrogen phosphates, the calcium chloride of 0.05-0.1g, the magnesium chloride of 0.1g.
8. the preparation method of mice embryonic according to claim 7, it is characterised in that the embryonic cell screens step
In, embryo's mixed liquor also contains based on per liter：The calf serum of the bovine serum albumin solution or 10-100ml of 3-6ml；
The penicillin of ten thousand units of 60-85, the kanamycin of ten thousand units of 60-85.
9. a kind of mouse embryo cell, it is characterised in that system of the mouse embryo cell by described in any one of claim 1-6
Preparation Method is obtained.
10. the No operation transfer method of a kind of mouse embryo cell, it is characterised in that by Development of Mouse Embryos as claimed in claim 9
Fetus cells are developed to blastula stage, and the mouse embryo cell that will be in the blastula stage loads grafts, and by the transplanting
Pipe is put in embryo transfer gun, and the embryo transfer gun is sent into the intrauterine of recipient female mice, and insertion transplanting rifle inner core will
The mouse embryo cell sends into the intrauterine of the recipient female mice.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798567A (en) * | 2009-02-11 | 2010-08-11 | 北京华盛兴邦生物技术有限公司 | Modified culture method for promoting cloned mouse embryonic development |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101798567A (en) * | 2009-02-11 | 2010-08-11 | 北京华盛兴邦生物技术有限公司 | Modified culture method for promoting cloned mouse embryonic development |
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