CN104513807B - The method for cloning non-human animal is separated, cultivates the method for cell and carried out from blood - Google Patents
The method for cloning non-human animal is separated, cultivates the method for cell and carried out from blood Download PDFInfo
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Abstract
The invention provides a kind of separation from blood, the method for cultivating cell and a kind of method cloned to non-human animal.The method for being separated from blood, cultivating cell comprises the following steps：The blood is added to behind separating liquid upper strata and centrifuged using the mode of density gradient centrifugation, to obtain mononuclearcell；The mononuclearcell is inoculated in the in vitro culture equipment containing attached cell culture medium, to obtain attached cell；And the attached cell is subjected to Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting.The donorcells for the body-cell neucleus transplanting that this method obtains is used directly for handmade cloning non-human animal, not only operating process is simple, be easily achieved, cost is relatively low, and this method is stimulated less Animal stress, injury is small, it is ensured that the integral outer appearance of animal, improves animal welfare.
Description
Technical field
The present invention relates to biology field, in particular it relates to which method and the clone of cell are separated, cultivated from blood
The method of non-human animal.
Background technology
Body-cell neucleus transplanting is also known as cloned, and is the most frequently used technological means of System in Animal Cell Biotechnology.By electric shocking method or directly
The higher body cell of differentiation degree is transferred in egg mother cell by microinjection, then by chemokinesis, reprograms it
Embryo is developed into, is transplanted back in foster mothers, complete individuals of being born.Traditional somatic cell nuclear transfer technique need it is expensive,
Accurate micromanipulation system, it is very high to the technical requirements of operating personnel, it is not suitable for large-scale cloned animal production.
2001, Danish scientist Gabor Vajta proposed handmade cloning technology first, the maximum key point of the technology
It is to utilize blade to realize " cutting stoning ", so as to be completely free of micromanipulation system, reduces the instrument throwing for cloning this technology
Enter and artificial expenditure cost, make it possible that this technology of clone is applied to following industrialization.Body-cell neucleus transplanting at present
Blastocyst rate between 20%-30%, and handmade cloning animal blastocyst rate can be improved to 40%-60%.
Thus, the donor cell source currently used for somatic cell cloning still has much room for improvement.
The content of the invention
It is contemplated that at least solves one of above-mentioned technical problem to a certain extent.
The present invention is inventor based on the fact that finding：In recent years, cloned animal increasingly have its it is wide should
With prospect, such as the cultivation available for high-quality poultry kind, to be advantageous to animals on the brink of extinction Germ-plasma resources protection；All kinds of experiments are produced to move
Thing, being derived from experimental animal has the characteristics that inhereditary material homogeneity, phenotype uniformity, is more suitable for pharmacology items and grinds
Study carefully；Clone is applied into transgenosis, can high efficiency obtain transgenic animals, shorten experimental period, largely obtain transgenic animals.
During body-cell neucleus transplanting, donorcells species is various, including is filled between the embryonic stem cell with multipotency, fat
Matter stem cell, and terminal differentiation cell fibroblast, liver cell, granular cell, liver cell etc..Derived from embryonic stem cells
In the inner cell mass of blastula stage, generations up to a hundred can be exceeded by being cultivated on feeder cells, but cumbersome, be unsuitable for building on a large scale
System's passage, and due to the particularity in its source, the relevant information of adult animal can not be obtained, clone has for high-quality germline
Certain limitation；And the mescenchymal stem cell attached cell in bone source, mainly by the marrow in separating animal's femur and
Target cell is obtained, but the sample for using the method to collect is acquired, it is necessary to which animal is first put to death to facilitate；Other sources
For donor cell, tissue, organ wounds can be all caused to animal individual to varying degrees.It is at present the most normal into fiber
Clone's cell somatic types, but for the big animal such as pig, ox, sheep, it is substantially and gathers its ear-edge tissue and carry out in vitro
Original cuiture, but the reason such as growing environment factor due to itself, sample collection it is difficult to ensure that sterile, train in vitro by the later stage
It is very high to support bacterium, the probability of fungal contamination in preparation process.
Therefore, it is an object of the present invention to propose that a kind of stress stimulation to animal is few, injury is small, ensure outside animal
See the method for completely being separated from blood, cultivating cell.This method comprises the following steps：The blood is added to separating liquid
Centrifuged behind upper strata using the mode of density gradient centrifugation, to obtain mononuclearcell；The mononuclearcell is inoculated with
In the in vitro culture equipment containing attached cell culture medium, to obtain attached cell；And the attached cell is passed
It is commissioned to train foster, to obtain the donorcells of body-cell neucleus transplanting.By the extraction according to embodiments of the present invention from blood, cultivate
The donorcells of body-cell neucleus transplanting prepared by the method for cell is used for handmade cloning body-cell neucleus transplanting, and blastaea efficiency is in highest
Up to 55%, average blastocyst rate is 40% or so.Thus, the side for being separated from blood, cultivating cell according to embodiments of the present invention
Not only operating process is simple for method, be easily achieved, cost is relatively low, and this method is stimulated less Animal stress, injury is small, it is ensured that
The integral outer appearance of animal, improve animal welfare.
In addition, the method for being separated from blood, cultivating cell according to embodiments of the present invention can also be with following additional
Technical characteristic：
According to an embodiment of the invention, the blood comes from peripheral blood.Due to peripheral blood acquisition operations are relatively easy, to dynamic
Thing stress stimulation is few, injury is small, thus can not only improve animal welfare, and blood is comparatively closed with the external world, and the later stage is thin
Born of the same parents' culture pollution rate is substantially zeroed, improves cell survival rate.
According to an embodiment of the invention, the separating liquid is neutrophil leucocyte separating liquid.Thus, it is possible to effectively pass through profit
The various cell components in blood are separated according to the size of density with the mode of density gradient centrifugation, by centrifugally operated
If blood is divided into dried layer, contain different blood cell components in every layer, so as to effectively obtain mononuclearcell.
According to an embodiment of the invention, the density of the neutrophil leucocyte separating liquid is 1.077g/mL, wherein, in described
Property granulocyte separating liquid contains 57g/L ficolls and 90g/L Sodium Amidotrizoates.Thus, the neutrophil leucocyte containing mentioned component point
Chaotropic improves the separative efficiency of different cell components in separation blood, so as to which effectively mononuclear cell layer obtains therefrom
Mononuclearcell in blood.
According to an embodiment of the invention, the mononuclearcell is collected from intermediate layer after separating liquid centrifugation.Profit
Blood added in above-mentioned separating liquid with the mode of density gradient centrifugation after being centrifuged, blood quilt in above-mentioned separating liquid
If being divided into dried layer, contain different cell components in every layer respectively, thus, it is possible to directly collect mononuclearcell from intermediate layer, no
Only experimental implementation process is simple, is easy to test, and the cell component in every layer after layering is single, and it is thin to which thereby enhance single core
The separative efficiency of born of the same parents.
According to an embodiment of the invention, the mononuclearcell is inoculated in the in vitro culture containing attached cell culture medium
Equipment, further comprise to obtain attached cell：First, will be adherent thin using first with the mononuclearcell after PBS
Born of the same parents' culture medium is cultivated 48 hours in 37 degrees Celsius of CO2gas incubator, first attached cell culture include low sugar-
MEM, 15%FBS, 1 × nonessential amino acid, 10ng/mL bFGF and 30IU/mL heparin；Then, culture medium is replaced by second
Attached cell culture medium continues to cultivate in 37 degrees Celsius of CO2gas incubator, and the second attached cell culture medium includes
MEM- α, 15%FBS, 1 × nonessential amino acid and 10ng/mL bFGF.Thus, can be first by the mononuclearcell of separation
Preliminary adaptability in vitro culture is carried out under suitable nutritional condition, the mononuclearcell is by the first patch containing low sugar-MEM
After parietal cell medium culture 48 hours, culture medium is replaced by the second attached cell culture medium containing MEM- α and continues to cultivate,
So as to obtain the good cell of adherence quality, and to grow up under microscope in spindle shape, its nucleus is larger, and growth is very fast,
Therefore it is suitable for the experiment of handmade cloning body-cell neucleus transplanting.
According to an embodiment of the invention, the in vitro culture equipment is the coated culture dish of gelatin or the coated culture of gelatin
Plate.Because gelatin is the major structural components of the extracellular matrix of multicellular organism, can promote the adherent of cell and increase
Grow, specific morphological and functional expression, be usually used in the in vitro culture of the primary cell of various cells.Thus list can not only be improved
Adherent efficiency of the individual nucleus in culture device, and compared with other adherent class reagents of rush, experimental cost is relatively low.
According to an embodiment of the invention, attached cell progress Secondary Culture is further comprised, will be described adherent thin
Born of the same parents' Secondary Culture 2-20 generations.Thus, it is possible to it will enter in exponential phase, the mononuclearcell that activity is good, nucleus is larger
One step is tested for handmade cloning body-cell neucleus transplanting.
Another object of the present invention is to provide a kind of method cloned to non-human animal, including：Using it is above-mentioned from
The method for being separated in blood, cultivating cell, prepare the donorcells of somatic cell cloning；And based on the body-cell neucleus transplanting
Donorcells, by handmade cloning, carry out clone non-human animal.Thus, it is possible to using it is according to embodiments of the present invention it is above-mentioned from
The cell for separating, cultivating in blood, by handmade cloning, obtain non-human animal.Due to by it is according to embodiments of the present invention from
The method of cell is separated, cultivated in blood, and not only experimental implementation process is simple, be easily achieved, cost is relatively low, and this method is to dynamic
Thing stress stimulation is few, injury is small, it is ensured that the integral outer appearance of animal, improve animal welfare, therefore by the somatic cell nuclear of acquisition
The donorcells of transplanting is used for non-human animal clone and had broad application prospects.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 is the method schematic diagram for being separated from blood, cultivating cell according to the embodiment of the present invention.
Fig. 2 is the side that non-human cell clone is carried out using the cell being separately cultured from blood according to the embodiment of the present invention
Method schematic diagram.
Fig. 3, which is shown, to be cultivated after pig vena cava anterior density gradient separation according to embodiments of the present invention after 7d by 200 times
The cytological map that phase contrast microscope is observed.
Fig. 4, which is shown, to be cultivated after pig vena cava anterior density gradient separation according to embodiments of the present invention after 7d by 200 times
The cytological map that phase contrast microscope is observed, which part cell grow in contact inhibition.
Fig. 5 show it is according to embodiments of the present invention from blood separate cell carry out adhere-wall culture be passaged to P1 generation pass through
The cytological map that 100 times of phase contrast microscopes are observed.
Fig. 6 show it is according to embodiments of the present invention from blood separate cell carry out adhere-wall culture be passaged to P1 generation pass through
The cytological map that 200 times of phase contrast microscopes are observed.
Fig. 7：Show that the cell according to embodiments of the present invention that will be separated, cultivate is used for body-cell neucleus transplanting, the 5th day logical
Cross the blastaea aspect graph that 100 times of Huffman microscopes are observed.
Fig. 8：Show flow cytometric analysis results figure according to embodiments of the present invention：
8A is the surface markers fluidic cell result figure of CD29 egg mother cell；
8B is the surface markers fluidic cell result figure of CD90 egg mother cell；
8C is the surface markers fluidic cell result figure of CD34 egg mother cell；
8D is the surface markers fluidic cell result figure of CD45 egg mother cell.
Fig. 9 is the donorcells according to body-cell neucleus transplanting of the method based on the embodiment of the present invention of the embodiment of the present invention,
And the non-human animal obtained by handmade cloning.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
In addition, term " first ", " second " are only used for describing purpose, and it is not intended that instruction or hint relative importance
Or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can be expressed or
Implicitly include one or more this feature.In the description of the invention, " multiple " are meant that two or more,
Unless otherwise specifically defined.
The present invention is inventor based on the fact that finding：In recent years, cloned animal increasingly have its it is wide should
With prospect, such as the cultivation available for high-quality poultry kind, to be advantageous to animals on the brink of extinction Germ-plasma resources protection；All kinds of experiments are produced to move
Thing, being derived from experimental animal has the characteristics that inhereditary material homogeneity, phenotype uniformity, is more suitable for pharmacology items and grinds
Study carefully；Clone is applied into transgenosis, can high efficiency obtain transgenic animals, shorten experimental period, largely obtain transgenic animals.
During body-cell neucleus transplanting, donorcells species is various, including is filled between the embryonic stem cell with multipotency, fat
Matter stem cell, and terminal differentiation cell fibroblast, liver cell, granular cell, liver cell etc..Derived from embryonic stem cells
In the inner cell mass of blastula stage, generations up to a hundred can be exceeded by being cultivated on feeder cells, but cumbersome, be unsuitable for building on a large scale
System's passage, and due to the particularity in its source, the relevant information of adult animal can not be obtained, clone and have for high-quality germline
There is certain limitation；And the mescenchymal stem cell attached cell in bone source, mainly pass through the marrow in separating animal's femur
And target cell is obtained, but the sample for using the method to collect is acquired, it is necessary to which animal is first put to death with facilitating；Other sources
Be used for donor cell, tissue, organ wounds can be all caused to animal individual to varying degrees.It is at present the most normal into fiber
Clone's cell somatic types, but for the big animal such as pig, ox, sheep, it is substantially and gathers its ear-edge tissue and carry out in vitro
Original cuiture, but the reason such as growing environment factor due to itself, sample collection it is difficult to ensure that sterile, train in vitro by the later stage
It is very high to support bacterium, the probability of fungal contamination in preparation process.
Thus in the first aspect of the invention, the present invention proposes a kind of method for being separated from blood, cultivating cell.
This method comprises the following steps：By the blood be added to behind separating liquid upper strata using the mode of density gradient centrifugation carry out from
The heart, to obtain mononuclearcell；The mononuclearcell is inoculated in the in vitro culture equipment containing attached cell culture medium,
To obtain attached cell；And the attached cell is subjected to Secondary Culture, it is thin to obtain the donor of body-cell neucleus transplanting
Born of the same parents.The donor of the body-cell neucleus transplanting prepared by the method according to embodiments of the present invention that cell is extracted, cultivated from blood
Cell is used for handmade cloning body-cell neucleus transplanting, and blastaea efficiency is reaching as high as 55%, and average blastocyst rate is 40% or so.Thus,
Not only operating process is simple for the method for being separated from blood, cultivating cell according to embodiments of the present invention, be easily achieved, cost compared with
It is low, and this method is stimulated less Animal stress, injury is small, it is ensured that the integral outer appearance of animal, improve animal welfare.
Below in conjunction with the accompanying drawings 1 and embodiment to the present invention from blood separate, cultivate cell method do into
One step describes in detail：
S101：Blood is added to behind separating liquid upper strata and centrifuged using the mode of density gradient centrifugation, to obtain
Mononuclearcell
According to an embodiment of the invention, the mode for separating cell in blood is not particularly restricted, as long as can reach institute
Using blood collection by the way of animal stimulated injure small purpose, blood collection method commonly used in the art can be used,
According to some embodiments of the invention, blood collection can be carried out by modes such as tail vein, auricular veins, according to the one of the present invention
Individual specific example, pig vena cava anterior blood mode can directly be extracted by using 50mL syringe from pig and extract periphery
Blood.Due to peripheral blood acquisition operations are relatively easy, to Animal stress stimulate less, injury it is small, thus can not only improve animal good fortune
Profit, and blood is comparatively closed with the external world, later stage pollution of cell culture rate is substantially zeroed, improves cell survival rate.
Embodiments of the invention, the mode preserved to the peripheral blood of above-mentioned acquisition are not particularly restricted, as long as can
The peripheral blood that collection from obtaining to prevent solidifies before separating step is carried out, and can use anti-freezing mode commonly used in the art, root
According to the specific embodiment of the present invention, it can use and heparin progress anti-freezing is added into the peripheral blood gathered.Inventor is surprised
It was found that 125IU heparin is added in the peripheral blood gathered to 10mL can effectively prevent gathered periphery blood clotting.This
Outside, according to an embodiment of the invention, the peripheral blood for adding heparin is placed in carrying out preserving transport under 4 degrees celsius.Thus,
Gathered periphery blood clotting can be not only prevented, and can prevent the effective active cell component in peripheral blood from degrading, from
And beneficial to the separation cell manipulation of next step.
Due to containing red blood cell, eosinophil, basophilic granulocyte, neutrophil leucocyte, T lymphocytes, B in blood
Lymphocyte, NK, macrophage, monocyte, granulophilocyte, thick liquid cell, mast cell, blood platelet,
The various cells such as mesenchymal cells, fibroblast, adipocyte and desmacyte, it is therefore desirable to above-mentioned cell is separated,
The donorcells for extracting body-cell neucleus transplanting therefrom is used, so as to be used for handmade cloning.According to an embodiment of the invention, from blood
The mode of middle separation mononuclearcell is not particularly restricted, and can use various cell separate modes commonly used in the art, root
According to the specific example of the present invention, it can utilize and above-mentioned gathered peripheral blood is added into separating liquid upper strata and density gradient centrifugation
Method separates to above-mentioned peripheral blood.Thus, it is possible to the mode of separating liquid and density gradient centrifugation is effectively utilized by blood
Various cell components in liquid are separated according to the size of density, if being divided into dried layer by centrifugally operated blood, in every layer
Containing different blood cell components, so as to effectively obtain mononuclearcell.
According to an embodiment of the invention, the used separating liquid separated to above-mentioned peripheral blood is not limited especially
System, according to the specific example of the present invention, the separating liquid is neutrophil leucocyte separating liquid, the neutrophil leucocyte separating liquid it is close
Spend for 1.077g/mL, and the specific composition for having the neutrophil leucocyte separating liquid is 57g/L ficolls and 90g/L amidotrizoic acids
Sodium.Thus, the neutrophil leucocyte separating liquid containing mentioned component improves the separative efficiency of different cell components in separation blood,
So as to effectively obtain the mononuclearcell in blood from the above-mentioned mononuclear cell layer by layering peripheral blood.According to this hair
Bright embodiment, if blood constituent after centrifugation is divided into dried layer according to different density, point in test tube from top to bottom
Layer includes but is not limited to plasma layer, cellular layer, separation liquid layer, neutrophil leucocyte layer, remaining separation liquid layer and erythroprecipitin
Layer, wherein the mononuclearcell is in middle Yunfu shape cellular layer.According to an embodiment of the invention, collect separated single
The method of nucleus is not particularly restricted, and can be various collection methods commonly used in the art, and specific according to the present invention is shown
Example, can draw out using transfer pipet(te) by the mononuclearcell in middle Yunfu shape cellular layer, and further to drawing out
The mononuclearcell come is cleaned, and according to a particular embodiment of the invention, the number of cleaning can be 2-3 times.Thus, it is possible to
The separative efficiency to blood cell is improved, so as to obtain the mononuclearcell of high quality.
S102：Mononuclearcell is inoculated in vitro culture equipment, to obtain attached cell
According to an embodiment of the invention, the above-mentioned mononuclearcell isolated and cleaned is inoculated in containing attached cell
The in vitro culture equipment of culture medium, to obtain attached cell.According to an embodiment of the invention, the mononuclearcell to isolating
The mode for carrying out in vitro culture is not particularly restricted, can be with as long as can cause the adherent normal growth of above-mentioned mononuclearcell
Using various extracorporeal culturing methods commonly used in the art, according to some embodiments of the present invention, first by through the single of over cleaning
Nucleus is cultivated 48 hours using the first attached cell culture medium in 37 degrees Celsius of CO2gas incubator.According to the present invention
Embodiment, the composition of the first attached cell culture medium is not particularly limited, as long as containing enough nutrition and being adapted to
The mononuclearcell just separated from blood carries out adherent growth in culture device in vitro, can use commonly used in the art
Various adhere-wall culture bases, according to a particular embodiment of the invention, the first attached cell culture medium can include low sugar-MEM,
15%FBS, [1 ×] nonessential amino acid, 10ng/mL bFGF and 30IU/mL heparin.According to some embodiments of the present invention, 48
The first attached cell culture medium is replaced by titanium dioxide of the second attached cell culture medium continuation at 37 degrees Celsius after hour
Cultivated in carbon incubator, the second attached cell culture medium volume can include MEM- α, 15%FBS, [1 ×] nonessential amino acid
With 10ng/mL bFGF.Wherein, according to an embodiment of the invention, the first attached cell culture medium is changed after 48 hours
Mode for the second attached cell culture medium is not particularly restricted, can be first by described the according to the specific example of the present invention
One attached cell culture medium discards, and then cell is cleaned using PBS to remove the culture of the first attached cell of residual
Base, then the second attached cell culture medium is added in adherent mononuclearcell and continues to cultivate, according to the present invention
Embodiment, the number of cleaning can be 1-2 times.Wherein, MEM- α are a kind of MEM culture mediums of improvement, with the addition of it is a variety of it is non-must
Need amino acid and Anacobin, C etc.；And can promote cell attachment using low sugar-MEM, using MEM- α after 48h instead can be reduced
Adherent haemocyte, so as to be advantageous to adherent karyocyte growth.Thus, the mononuclearcell by separation can exist first
Preliminary adaptability in vitro culture is carried out under suitable nutritional condition（Utilize the first attached cell culture medium containing low sugar-MEM
Culture 48 hours）, culture medium is replaced by be more suitable for the second attached cell culture medium containing MEM- α of adherent growth after
Continuous culture, so that the above-mentioned mononuclearcell separated from peripheral blood can form suitable and abundance in nutritional ingredient
Adhere-wall culture base in carry out appropriate growth.
According to an embodiment of the invention, used in vitro culture equipment is not particularly restricted, as long as being adapted to from blood
In the mononuclearcell growth isolated, various culture devices commonly used in the art can be used and be selected as needed,
Such as 35mm culture dishes, 60mm culture dishes, 100mm culture dishes, 6 orifice plates and 4 orifice plates, according to the specific example of the present invention, adopted
In vitro culture can be the coated culture dish of gelatin or the coated culture plate of gelatin.Because gelatin is multicellular organism
Extracellular matrix major structural components, the adherent of cell and propagation, specific morphological and functional expression can be promoted, commonly used
In the in vitro culture of the primary cell of various cells.Thus adherent effect of the mononuclearcell in culture device can not only be improved
Rate, and reduce experimental cost.According to an embodiment of the invention, using and attached cell culture medium to adherent single core
The mode that cell is cultivated is not particularly restricted, and according to the specific example of the present invention, can once be changed using 2-3 days
Liquid is operated so as to keep adherent mononuclearcell to be in the suitable and sufficient attached cell culture medium of nutritional ingredient all the time
In grown.The mononuclearcell adherence quality that method according to embodiments of the present invention obtains is good, and growth is very fast, under microscope
Grow up in spindle shape, its entoblast is larger and high-visible, therefore is suitable for handmade cloning body-cell neucleus transplanting（See figure
3）.
S103：Attached cell is subjected to Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting
According to an embodiment of the invention, when adherent mononuclearcell fills into bulk contact inhibition to be grown（See Fig. 4）, can
To carry out passage culture., can be by the attached cell Secondary Culture 2-20 generations according to the specific example of the present invention.According to
Embodiments of the invention, by above-mentioned in a manner of bulk contact inhibition fills the adherent mononuclearcell progress Secondary Culture of growth
It is not particularly restricted, can is various propagating methods commonly used in the art, according to the specific example of the present invention, can uses such as
Lower step：Discard former culture medium；Add PBS rinse cell 2 times；Cell dissociation buffer is added after discarding PBS
Cell, and cell is positioned in 37 degrees Celsius of CO2gas incubator and digested；Add adhere-wall culture base 2 and with warp
The mononuclearcell for crossing digestion is well mixed, to obtain cell suspension；Above-mentioned cell suspension is transferred to and is coated with by gelatin
Culture device in carry out Secondary Culture.Mononuclearcell is obtained by the method for the cultured cell line of the embodiment of the present invention to give birth to
Length is vigorous, and proliferative is good（See Fig. 5 and Fig. 6）.Wherein, according to an embodiment of the invention, used digestive juice is not by special
Limitation, if under the microscope observation can cause attached cell is gradually rounded to brighten, and progressively disengage cell cultivation equipment from
And float in cell dissociation buffer, various cell dissociation buffers commonly used in the art can be used, according to the specific implementation of the present invention
Example, cell dissociation buffer can be 0.05% pancreatin -200mg/L EDTA.Thus, it is possible to will be in exponential phase, activity it is good,
The larger mononuclearcell of nucleus is further used for the experiment of handmade cloning body-cell neucleus transplanting.
In another aspect of this invention, the invention provides a kind of method cloned to non-human animal, including：Utilize
The above-mentioned method for being separated from blood, cultivating cell, prepare the donorcells of somatic cell cloning；And based on the somatic cell nuclear
The donorcells of transplanting, by handmade cloning, carry out clone non-human animal.Thus, it is possible to utilize according to embodiments of the present invention
The above-mentioned cell for separating, cultivating from blood, by handmade cloning, obtain non-human animal.Due to by being implemented according to the present invention
Not only experimental implementation process is simple for the method for being separated from blood, cultivating cell of example, be easily achieved, cost is relatively low, and the party
Method is stimulated less Animal stress, injury is small, it is ensured that the integral outer appearance of animal, improve animal welfare, therefore by the body of acquisition
The donorcells of nuclear transplantation is used for non-human animal clone and had broad application prospects.
Below in conjunction with the accompanying drawings 2 and embodiment the present invention is carried out using the cell that is separately cultured from blood it is non-
The method of people's cell clone is described in further details：
S201：Oocyte IVM and surface molecular identification
According to an embodiment of the invention, the mode of oocyte collection is not particularly restricted, as long as health can be obtained
Egg cell, can use various methods commonly used in the art, according to a particular embodiment of the invention, following step can be included
Suddenly：The ovary of collection is placed in the container equipped with physiological saline and keeps 35 degrees Celsius of constant temperature；Then above-mentioned ovary is put
It is placed in the culture dish equipped with embryo's cleaning solution, is scratched ovarian follicle using blade, so that egg cell flows out；And will be above-mentioned
Egg cell is collected in the culture device containing egg cell culture medium of pre-balance and cultivated to egg maturation.According to the present invention
Embodiment, egg cell culture to the condition of maturation is not particularly limited, can be that various egg cells commonly used in the art are trained
The technology of supporting, according to the specific example of the present invention, in order to reach the metastable culture environment of pH value, culture medium is placed in advance
The CO2gas incubator of 5% concentration is balanced, and then contains embryo maturation culture medium by what egg cell was collected in pre-balance
（IVM）In four orifice plates, each 50 embryos in hole, and the above-mentioned four hole versions for being loaded with egg cell are placed in 38.5 degrees Celsius of titanium dioxide
Ripe 42h in carbon incubator.According to an embodiment of the invention, above-mentioned embryo's cleaning solution is not particularly restricted, and can be ability
The conventional various liquid washed to embryo in domain, according to the specific example of the present invention, used embryo's cleaning solution（ASP）
In include：10%v/v 10 × TCM199,0.17mg/mL sodium acid carbonate, 0.20mg/mL Sodium Pyruvate, 3.60mg/mL are conspicuous
Wear this sodium salt, 2.63mg/mL He Peisi acid, 0.20mg/mL glutamine, 3.00 μ g/mL amphotericin B, 30IU/mL
Heparin, 2% serum.Thus, it is possible to the fragment in ovary cut substrate is removed, so as to reduce the pollution of cultivated aim cell
Probability, improve cell survival rate.
According to an embodiment of the invention, embryo maturation culture medium（IVM）It is not particularly restricted, can is commonly used in the art
The culture medium cultivated embryo, according to the present invention instantiation, used embryo maturation culture medium（IVM）Bag
Contain：TCM199, the 5IU/mL of [1 ×] human chorionic gonadotrophin, 1IU/mL pregnant mare serum, 0.10mg/mL paddy ammonia
Acid amides, 0.05mg/mL gentamicins, 10% liquor folliculi, 10%v/v serum.Thus, passed through by making egg cell in the culture medium
The culture of 42 hours, the maturation of egg mother cell can be promoted, first polar body be discharged, so as to improve into heat efficiency（First polar body is arranged
Extracting rate）, approximately more than 80%.
According to an embodiment of the invention, the method identified the egg mother cell obtained is not particularly limited, can be with
For various cellular identification methods commonly used in the art, including but not limited to cell surface molecule labelling method, flow cytometry etc..Root
According to the specific embodiment of the present invention, CD29, CD90, CD34, CD45, FITC Mouse IgG1 and PE Mouse IgG17 are utilized
Kind different antibody carries out surface markers to the egg mother cell obtained, then using BD FACSCalibur flow cytometers with
And WinMDI2.8 softwares are analyzed, to determine whether obtained cell is mescenchymal stem cell.
S202：Egg mother cell takes off ovarian cumulus and stoning
By the ripe egg cell of above-mentioned culture from containing embryo maturation culture medium（IVM）Four orifice plates in be transferred to hyalomitome
In sour enzyme, to slough cumulus granulosa cells.According to an embodiment of the invention, will cultivate ripe egg cell from containing embryo into
Ripe culture medium（IVM）Four orifice plates in the transfer operation that is transferred in hyaluronidase be not particularly restricted, can be ability
The technology of the conventional transfer egg cell in domain, it is according to a particular embodiment of the invention, using liquid-transfering gun that ripe complete ovum is female thin
Born of the same parents are from containing embryo maturation culture medium（IVM）Four orifice plates in into hyaluronidase.Wherein, according to an embodiment of the invention,
The concentration of used hyaluronidase is not particularly restricted, as long as cumulus granulosa cells, people in the art can be sloughed
Member can carry out appropriate regulation according to the concentration of cell and the species of reagent, according to a particular embodiment of the invention, be adopted
The concentration of hyaluronidase can be 1mg/mL.Hyaluronidase main function is hyaluronic acid between dissolving cumulus cell,
So that cumulus cell disperses, thus, it is possible to using appropriate concentration by the cumulus cell in the ripe egg cell of above-mentioned culture
Disperse further to remove.According to an embodiment of the invention, the operation for sloughing cumulus granulosa cells is not particularly restricted, can
To use various technologies commonly used in the art, according to the specific implementation of present invention profit, inventor has surprisingly found that, using with rifle
The liquid-transfering gun of head carrys out resorption and beats the hyaluronidase containing egg mother cell, egg cell can be entered repeatedly using the small-bore of pipette tips
Row, which is inhaled, to be beaten, so as to slough the cumulus granulosa cells that mature egg pericellular carries out being metabolized therewith contact.
According to an embodiment of the invention, the method for removing ootid nucleus is not particularly restricted, and can be commonly used in the art
Various pitting methods, according to the present invention instantiation, can be first by the above-mentioned exposed egg mother cell for eliminating cumulus cell
It is positioned in protease, it is loose so as to which the oolemma on egg mother cell top layer is digested, it is then transferred to the second embryo operation
In liquid operation drop, and the method positioned according to polar body, first polar body is cut off together with part egg mother cell matter.According to the present invention's
Embodiment, the second embryo operation liquid can the sodium acid carbonate including 10%v/v [10 ×] TCM199,0.17mg/mL, 0.20
Mg/mL Sodium Pyruvate, 3.60mg/mL He Peisi sodium salts, 2.63mg/mL He Peisi acid, 0.20mg/mL glutamy
Amine, 2.5 μ g/mL B cytochalasin Bs and 20% serum.Thus, it is possible to effectively remove oocyte nuclei.
According to an embodiment of the invention, placement is above-mentioned is not particularly restricted by the mode of non-nucleus egg mother cell, this
Art personnel can need to be selected according to subsequent operation, according to the specific example of the present invention, by the ovum of above-mentioned stoning
Mother cell is transferred to stand-by in the first embryo operation liquid operation drop.According to an embodiment of the invention, the first embryo operation liquid can be with
[10 ×] TCM199,0.17mg/mL sodium acid carbonate, 0.20mg/mL Sodium Pyruvate, 3.60mg/mL including 10%v/v
He Peisi acid, 0.20mg/mL glutamine, 2% serum of He Peisi sodium salts, 2.63mg/mL.Thus, the process obtained
Non-nucleus egg mother cell is survived in containing most suitable embryo operation pendular ring border.
S203：Egg mother cell merges with donorcells
According to an embodiment of the invention, the body cell obtained in S103 is scattered in into above-mentioned first embryo operation liquid first to grasp
In dripping, thus, body cell is in the first embryo operation liquid operation drop in individual cells distribution shape；Then will be obtained in S202
Above-mentioned the first embryo containing body cell is transferred to after non-nucleus egg mother cell places a period of time in phytolectin
In tire operation liquid operation drop, wherein according to some instantiations of the present invention, it is female thin by the ovum being enucleated by being obtained in S202
Born of the same parents place 1~2 second in 1mg/mL phytolectins, it is possible thereby to make cytosolic surface band toughness, so that single body is thin
Born of the same parents and process non-nucleus egg mother cell are sufficiently bonded in the first embryo operation liquid operation drop.
According to an embodiment of the invention, by non-nucleus egg mother cell（Kytoplasm）The method merged with donorcells is not
It is particularly limited, can is various cell-fusion techniques commonly used in the art, can be by born of the same parents according to the specific example of the present invention
Matter-body cell, which is transferred to, to be loaded with the integration slot of mixing operation liquid, is merged by electric shock.According to an embodiment of the invention,
Mixing operation liquid in integration slot can include 3M mannitol, 1mg/mL polyvinyl alcohol and 0.1mM magnesium sulfate.Thus egg mother cell
It may be in most suitable environmental condition receiving electric shock with body cell, so as to improve fusion efficiencies.According to the implementation of the present invention
Example, the condition merged to egg mother cell-body cell are not particularly restricted, and those skilled in the art can be according to specific thin
Born of the same parents' situation and equipment situation are adjusted, according to an embodiment of the invention, the bar that electric shock egg mother cell is merged with body cell
Part can be 100V direct currents electric shock 9 μ s, 4V alternating currents electric shock.It can depolarize kytoplasm by using alternating current, so as to
It is set to hang on wire electrode, and by electric pulse, cell membrane can be made to form an instantaneous perforation, so that kytoplasm
With donorcells film combination and merging.Thus, kytoplasm can receive electric shock with body cell under conditions of being best suitable for, so as to obtain
Sufficiently fusion, to obtain reconstruct clone embryos.According to an embodiment of the invention, the cell to have shocked by electricity is transferred to embryo behaviour
Make in the operation drop of liquid 1, and operation panel is placed in 38.5 degrees Celsius of thermal station and waited 1 hour, so as to improve late embryogenesis development
Efficiency.
S204：Reconstructed embryo is activated, forms embryo
According to an embodiment of the invention, it will be transferred to by the kytoplasm for having body cell that melts obtained in S203 steps and be loaded with activation
In the integration slot for operating liquid, and stoning ovum mother's kytoplasm obtained in S202 steps is hung, then shocked by electricity.Wherein utilize
Alternating current depolarizes reconstructed embryo so that it can be hung on wire electrode, second kytoplasm be also by unpolarizing, can
So that it can be contacted with reconstructed embryo.Pass through the above method so that an individual cells have merged two kytoplasms, and the composition of kytoplasm increases
Add, more nutriments can be provided, be advantageous to the embryonic development in later stage.According to an embodiment of the invention, above-mentioned activation manipulation
Liquid can be 3M mannitol, 1mg/mL polyvinyl alcohol, 0.1mM magnesium sulfate and 0.05mM calcium chloride.
According to an embodiment of the invention, the condition to be shocked by electricity is not particularly restricted, and those skilled in the art can root
It is adjusted according to specific cell situation and equipment situation, according to the instantiation of the present invention, the condition of electric shock can be 43V direct currents
Electricity 80 μ s of electric shock, are then shocked by electricity using 4V alternating currents.Thus, it can depolarize reconstructed embryo by using alternating current, so as to
It is set to hang on wire electrode, and by electric pulse, cell membrane can be made to form an instantaneous perforation, so that reconstruct
Embryo and cytoplasmic cell film combination simultaneously merge；Then the embryo transfer after electric shock is treated two into the first embryo operation liquid operation drop
Individual cytomixis.Thus, it is possible to improve the stability of embryo.
According to an embodiment of the invention, the mode for entering line activating to the above-mentioned cell by fusion is unrestricted, can
Think various activation techniques commonly used in the art, according to a particular embodiment of the invention, the embryo by fusion is subjected to chemistry
Activation, wherein according to the instantiation of the present invention, chemokinesis is carried out during the embryo transfer by fusion to chemokinesis is dripped.
According to some embodiments of the present invention, the above-mentioned chemically sensitized chemokinesis drop of carry out contains embryo culture medium（IVC）And 5 μ
G/mL B cytochalasin B, 10 μ g/mL cycloheximide.According to an embodiment of the invention, embryo culture medium（IVC）It can include
6.31mg/mL sodium chloride, 2.11mg/mL sodium acid carbonate, 0.75mg/mL potassium chloride, 0.05mg/mL biphosphate
The meso of potassium, 0.05mg/mL magnesium sulfate, 0.62mg/mL calcium lactate, 0.02mg/mL Sodium Pyruvate, 0.50mg/mL
Inositol, 0.01g/mL phenol red, 0.05mg/mL glutamine, 0.55mg/mL sub- ox acid iodide, 0.04mg/mL celebrating are mould greatly
Element, 3g/L bovine serum albumin(BSA), the nonessential amino acid and the essential amino acid of [0.5 ×] of [1 ×].It is possible thereby on so that
The cell by fusion is stated to be activated in most suitable medium component.According to other embodiments of the present invention, will pass through
It is in 38.5 degrees Celsius, 5% carbon dioxide and 5% oxygen culture that the embryo transfer of fusion, which carries out chemokinesis into embryo culture medium,
Carried out in case.According to the specific example of the present invention, taken the photograph by the embryo transfer by fusion into embryo culture medium and 38.5
Cultivated 4 hours in family name's degree, 5% carbon dioxide and 5% oxygen incubator.Thus, it is possible to so that the above-mentioned cell by activation is most
It is adapted to carry out adaptability growth under the environmental condition of growth, so as to improve survival rate of embryo.
According to an embodiment of the invention, by the above-mentioned embryo grown by activation and adaptability from above-mentioned chemokinesis drips
It is transferred in four orifice plates containing embryo culture medium of punching, and in 38.5 degrees Celsius, 5% carbon dioxide and 5% oxygen incubator
Continue to cultivate 120h or 144h.By punching multiple lensless Fourier transform holography reconstructed embryos can be made to be cultivated in same fluid apertures,
It is each to have oneself independent developmenting space by oneself, and close information can occur in same fluid apertures and exchange, be advantageous to embryonic development.
Thus, the above-mentioned embryo by activation and adaptability growth further can be grown under suitable conditions and environment, from
And improve the survival rate of embryo.
S205：Embryonic limb bud cell
According to an embodiment of the invention, the above-mentioned Embryonic limb bud cell by vitro culture is treated into pregnant animal fallopian tubal or uterus
It is interior, clone non-human animal by enough pregnancy periods.According to an embodiment of the invention, the above-mentioned embryo by vitro culture is planted
Enter to treat that pregnant animal fallopian tubal or intrauterine mode are not particularly restricted, can be the Embryonic limb bud cell skill that this area usually uses
Art, according to a particular embodiment of the invention, the blastaea to 120h will be developed（See Fig. 7）Enter to treat the son of pregnant parent by operation transplantation
In the angle of palace.According to an embodiment of the invention, embryo develops 114 days or so in parent, passes through spontaneous labor or output of cutting open the belly
Non-human animal is cloned, birth animal sees Fig. 9.
Embodiment 1 obtains mononuclearcell from blood
In advance into 15mL conical centrifuge tube add 125IU heparin, and by concussion make its be uniformly distributed in taper from
The tube wall of heart pipe.Pig vena cava anterior blood mode is directly extracted by the peripheral blood of miniature pig about 12mL by using 50mL syringe
It is collected in the above-mentioned conical centrifuge tube for adding heparin 15mL.The conical centrifuge tube for being loaded with peripheral blood is placed in 4 degrees Celsius
Transport can laboratory in ice chest.
Two 15mL conical centrifuge tube separately is taken, and is separately added into 5mL neutrophil leucocyte separating liquid thereto（Contain
57g/L ficolls and 90g/L Sodium Amidotrizoates）Afterwards, using liquid-transfering gun carefully along 15mL conical centrifuge tube wall and press close to neutrality
Granulocyte separating liquid liquid level is slowly added to the 5mL anticoagulation cirumferential blood gathered, and ensure the anticoagulation cirumferential blood not with lower floor
Neutrophil leucocyte separating liquid mixes, then horizontal centrifugal 30 minutes under the conditions of 400g.
After centrifugation, intermediate layer Yunfu shape mononuclearcell is collected from two centrifuge tubes respectively using liquid-transfering gun, and merge and put
It is placed in new 50mL conical centrifuge tube.By 20mL PBS be added to the foregoing taper containing mononuclearcell 50mL from
In heart pipe, and blown and beaten and cleaned repeatedly using Pasteur's dropper.After cleaning, the PBS suspensions containing mononuclearcell are existed
Centrifuged 10 minutes under the conditions of 300g.After centrifugation, 50mL conical centrifuge tube is taken out, remove after supernatant again to thin containing single core
20mL PBS is added in the 50mL of born of the same parents' precipitation conical centrifuge tube, and reuses Pasteur's dropper and blows and beats second of progress repeatedly
Cleaning.After second is cleaned, the PBS suspensions containing mononuclearcell are centrifuged 10 minutes under the conditions of 300g again.Then
Above-mentioned second of cleaning step is repeated, third time cleaning is carried out to mononuclearcell.
The mononuclearcell in vitro culture of embodiment 2
What is obtained into embodiment 1 adds 2mL after 3 times are cleaned and discarded and centrifuged in the cell precipitation of PBS supernatants
Containing low sugar-MEM, 15%FBS, [1 ×] nonessential amino acid, 10ng/mL bFGF and 30IU/mL heparin the first attached cell
Culture medium, and mononuclearcell precipitation piping and druming is dispersed in the first attached cell culture medium using Pasteur's dropper.It will contain
There is the first attached cell media transfer of mononuclearcell into the coated 35mm culture dishes of gelatin, it is Celsius to be then placed within 37
Cultivated in the CO2gas incubator of degree.
By the culture of 48 hours, taken out to the 35mm culture dishes for being loaded with mononuclearcell from incubator, discard first
After attached cell culture medium, add PBS and rinsed.After discarding PBS, added into the 35mm culture dishes for being loaded with mononuclearcell
The second attached cell culture medium containing MEM- α, 15%FBS, [1 ×] nonessential amino acid and 10ng/mL bFGF, and will culture
Ware puts back to incubator and continues to cultivate.And the 35mm culture dishes for being loaded with mononuclearcell were adopted respectively at the 72nd and 96 hour
Carried out changing liquid processing with the second attached cell culture medium.After mononuclearcell culture 5-7 days, light microscope progress is placed in
Observe, mononuclearcell is in into fibrous adherent cell growth on culture dish bottom surface under mirror（See Fig. 3）, and nucleus is larger,
Entoblast is high-visible.
The Secondary Culture of the mononuclearcell of embodiment 3
When adherent mononuclearcell is observed under the microscope fills growth in bulk contact inhibition（See Fig. 4）, discard training
Support the second attached cell culture medium in ware；Then PBS rinse cell is added 2 times；After discarding PBS, to being loaded with
200 μ L0.05% pancreatin -200mg/L EDTA are added in the culture dish of adherent mononuclearcell, and cell is positioned over 37 and taken the photograph
Digested in the CO2gas incubator of family name's degree, brighten and gradually take off when observing that attached cell is gradually rounded under the microscope
When being floated on from culture dish in cell pancreatin, 1mL the second attached cell culture mediums are added into culture dish, and drip by Pasteur
Pipe, which is blown and beaten, causes mononuclearcell to be well mixed with the fresh second attached cell culture medium added, to obtain cell suspension；
Then above-mentioned cell suspension is transferred to by carrying out Secondary Culture in the coated culture device of gelatin.Entered the patch of Secondary Culture
Wall mononuclearcell is observed under the microscope, and cell late growing stage is vigorous, and proliferative is good（See Fig. 5 and Fig. 6）.By Secondary Culture
2-20 be moved directly to for mononuclearcell cell suspension it is stand-by in 1.5mL centrifuge tubes, for handmade cloning.
The oocyte IVM of embodiment 4 and surface molecular identification
The pig ovary collected from slaughterhouse is placed in the container equipped with physiological saline to keep osmotic balance, and with
35 degrees Celsius of constant temperatures transport go back to laboratory.Ovary is positioned over equipped with embryo's cleaning solution（ASP）Culture dish in（Wherein embryo
Tire cleaning solution（ASP）The sodium acid carbonate of 10 × TCM199,0.17mg/mL containing 10%v/v, 0.20mg/mL Sodium Pyruvate,
3.60mg/mL He Peisi sodium salts, 2.63mg/mL He Peisi acid, 0.20mg/mL glutamine, 3.00 μ g/mL both sexes are mould
Plain B, 30IU/mL heparin, 2% serum）, ovarian follicle is scratched to cause the egg cell in ovarian follicle to flow out with blade.In order to reach
The metastable culture environment of pH value, the CO2gas incubator that culture medium is placed on to 5% concentration in advance are balanced, then
By the egg cell of outflow with the density collection of every 50 embryos in hole in the embryo maturation culture medium of pre-balance（IVM）Four orifice plates
In, wherein embryo maturation culture medium（IVM）Human chorionic gonadotrophin, the 1IU/mL of TCM199,5IU/mL containing [1 ×]
Pregnant mare serum, 0.10mg/mL glutamine, 0.05mg/mL gentamicins, 10% liquor folliculi, 10%v/v serum.Then will
Four orifice plates for being loaded with embryo are placed in 38.5 degrees Celsius of CO2gas incubators and cultivate 42h to maturation.
Adherent mature oocyte is subjected to digestion process, and centrifuged 3 minutes under the conditions of 350g, abandoning supernatant
Afterwards, 2mL PBS are added into precipitation and carries out piping and druming washing；Then again with 350g centrifuge 3 minutes, abandoning supernatant backward from
Precipitation in heart pipe adds 700 μ L PBS, and resulting suspension mean allocation is added into 7 new 2mL after piping and druming mixing
In centrifuge tube, i.e., often pipe adds 100 μ L cell suspending liquids；Then the μ L antibody 7 of 5 μ L~20 is added into each centrifuge tube respectively
Individual different antibody：CD29, CD90, CD34, CD45, FITC Mouse IgG1 and PE Mouse IgG1, vortex fully mix
Afterwards, lucifuge is incubated 15 minutes；Then 2 washings are carried out to the cell that marked surface antigen, condition is to add 1mL PBS,
Shake up and down it is even after with 350g centrifuge 3 minutes, abandon supernatant；Then again with 0.4mL poly methanol（4% concentration is dissolved in PBS）Dissolving washing
Cell precipitation afterwards, upper machine analysis is carried out to it using BD FACSCalibur, result is read using WinMDI2.8 softwares
Take, as a result referring to Fig. 8.Fig. 8 shows the high expression CD29 and CD90 surface antigens of cell according to embodiments of the present invention, without table
Up to CD34 and CD45 surface antigens.Fig. 8 result shows the high expression CD29 and CD90 surface antigens of obtained egg mother cell, no
CD34 and CD45 surface antigens are expressed, the CD29 and CD90 of the egg mother cell obtained in it positive rate are respectively 99.27% He
98.62, and CD34 and CD45 positive rate is respectively 0.43% and 0.45%.
The porcine oocytes of embodiment 5 take off ovarian cumulus and stoning
By the ripe egg cell liquid-transfering gun of above-mentioned culture from containing embryo maturation culture medium（IVM）Four orifice plates in shift
Into 1mg/mL hyaluronidases, with pipette tips come resorption since and slough cumulus granulosa cells.Then first ovum is eliminated by above-mentioned
The exposed egg mother cell of mound cell is positioned in protease, loose so as to which the oolemma for being covered in egg mother cell top layer is digested,
Then transfer them in the second embryo operation liquid operation drop, and the method positioned according to polar body, by first polar body together with part
Egg mother cell matter is cut off.Non-nucleus egg mother cell is transferred to it is stand-by in first embryo operation liquid operation drop, so as to be obtained
It can be survived by non-nucleus egg mother cell in containing most suitable embryo operation pendular ring border.Wherein the first embryo operation liquid can
With the sodium acid carbonate of [10 ×] TCM199,0.17mg/mL including 10%v/v, 0.20mg/mL Sodium Pyruvate, 3.60mg/mL
He Peisi sodium salts, 2.63mg/mL He Peisi acid, 0.20mg/mL glutamine, 2% serum.
The egg mother cell of embodiment 6 merges with donorcells
The body cell that embodiment 3 obtains is placed in the first embryo operation liquid operation drop, and causes body cell in the behaviour
In individual cells distribution shape in dripping.Pass through non-nucleus egg mother cell in 1mg/mL phytolectins by what embodiment 5 obtained
Place 1 second~2 seconds, the body cell then obtained with above-described embodiment 3, which merges to be put into foregoing first embryo operation liquid to operate, to drip
In, so that single body cell and process non-nucleus egg mother cell are sufficiently pasted in the first embryo operation liquid operation drop
Close.By enucleation oocyte（Kytoplasm）- body cell, which is transferred to, to be loaded with the integration slot of mixing operation liquid, is melted by electric shock
Close, to obtain reconstruct clone embryos.Mixing operation liquid wherein in integration slot include 3M mannitol, 1mg/mL polyvinyl alcohol and
0.1mM magnesium sulfate.The condition that electric shock egg mother cell is merged with body cell is 100V direct currents electric shock 9 μ s, 4V alternating currents electricity
Hit.Shocked by electricity all egg mother cells and body cell successively, transfers it in the first embryo operation liquid operation drop, will grasp
It is placed in 38.5 degrees Celsius of thermal station and waits 1 hour as disk.
Embodiment 7 activates reconstructed embryo, forms embryo
According to an embodiment of the invention, the kytoplasm that has body cell will be melted in embodiment 6 it is transferred to and is loaded with activation manipulation liquid
In integration slot（Wherein activation manipulation liquid contains 3M mannitol, 1mg/mL polyvinyl alcohol, 0.1mM magnesium sulfate and 0.05mM
Calcium chloride）, and the stoning kytoplasm obtained in embodiment 5 is hung, then shocked by electricity, to make reconstructed embryo and cytomixis：
Wherein depolarize reconstructed embryo using alternating current, so that it can be hung on wire electrode, second kytoplasm is also by going to pole
Change is acted on, and it can be made to be contacted with reconstructed embryo.The condition of electric shock is 43V direct currents 80 μ s of electric shock, then using 4V alternating currents
Electric shock, 4V alternating currents are until electric shock is closed after terminating.Thus, it can depolarize reconstructed embryo by using alternating current, so as to
So that it can be hung on wire electrode, and by electric pulse, cell membrane can be made to form an instantaneous perforation, so that weight
Structure embryo and cytoplasmic cell film combination simultaneously merge；Then the embryo transfer after electric shock is treated into the first embryo operation liquid operation drop
Two cytomixis.
Chemokinesis, the above-mentioned chemically sensitized chemistry of carry out are carried out during embryo transfer by fusion to chemokinesis is dripped
Activation drop contains embryo culture medium（IVC）And 5 μ g/mL B cytochalasin B and 10 μ g/mL cycloheximide.Wherein, embryo
Culture medium（IVC）The sodium acid carbonate of sodium chloride, 2.11mg/mL comprising 6.31mg/mL, 0.75mg/mL potassium chloride,
0.05mg/mL potassium dihydrogen phosphate, 0.05mg/mL magnesium sulfate, 0.62mg/mL calcium lactate, 0.02mg/mL pyruvic acid
Sodium, 0.50mg/mL meso inositol, 0.01g/mL phenol red, 0.05mg/mL glutamine, 0.55mg/mL sub- ox iodine
Acid, 0.04mg/mL gentamicin, 3g/L bovine serum albumin(BSA), [1 ×] nonessential amino acid and [0.5 ×] it is required
Amino acid.To through embryo transfer fusion into embryo culture medium in 38.5 degrees Celsius, 5% carbon dioxide and 5% oxygen culture
Chemokinesis is carried out in case, activationary time is 4 hours.
Punching is transferred to during the above-mentioned embryo by activation with adaptability growth is dripped from above-mentioned chemokinesis contains embryo
In four orifice plates of culture medium, and continue culture 120h in 38.5 degrees Celsius, 5% carbon dioxide and 5% oxygen incubator.
The Embryonic limb bud cell of embodiment 8
The above-mentioned Embryonic limb bud cell by vitro culture treats pregnant animal fallopian tubal or intrauterine, is cloned by enough pregnancy periods
Non-human animal.The blastaea to 120h will be developed first（See Fig. 7）Entered by operation transplantation in the cornua uteri for treating pregnant sow body.Embryo
Tire, by internal development 114 days or so, clones non-human animal, birth animal sees figure in sow by spontaneous labor or output of cutting open the belly
9。
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Claims (1)
- A kind of 1. method for the donorcells for being separated from blood, cultivating body-cell neucleus transplanting, it is characterised in that including following step Suddenly：(1) 12mL peripheral bloods are collected in the centrifuge tube equipped with 125IU heparin, mix, obtain anticoagulation cirumferential blood；(2) it is 1.077g/mL to the density equipped with 5mL, the neutrophil leucocyte containing 57g/L ficolls and 90g/L Sodium Amidotrizoates Anticoagulation cirumferential blood described in adding 5mL in separating liquid, ensures that neutrophil leucocyte separating liquid of the anticoagulation cirumferential blood not with lower floor is mixed Close, then horizontal centrifugal 30 minutes under the conditions of 400g, collect intermediate layer Yunfu shape mononuclearcell；(3) repeat step (2) once, and intermediate layer Yunfu shape mononuclearcell resulting twice is incorporated in centrifuge tube；(4) 20mL PBSs are added into step (3) described centrifuge tube, are purged, and 10 are centrifuged under the conditions of 300g Minute, collect precipitation；(5) by the operation 2 times of the precipitation repeat step (4), precipitation is collected, obtains mononuclearcell；(6) mononuclearcell is inoculated in containing low sugar-MEM, 15%FBS, 1 × nonessential amino acid, 10ng/mL First attached cell culture medium of bFGF and 30IU/mL heparin, and the first attached cell containing the mononuclearcell is trained Group-transfer is supported into the coated 35mm culture dishes of gelatin, is then placed within 37 degrees Celsius of CO2gas incubator and is trained Support；When culture to 48,72 and 96 hours when, by culture medium be replaced by containing MEM- α, 15%FBS, 1 × nonessential amino acid and 10ng/mL bFGF the second attached cell culture medium, continues to cultivate in 37 degrees Celsius of CO2gas incubator, culture 5 ~7 days, obtain attached cell；(7) attached cell 2 times described in PBS rinse are used, after discarding PBS, to the training for being loaded with the attached cell Support in ware and add the pancreatin -200mg/L EDTA of 200 μ L 0.05%, be placed in carrying out in 37 degrees Celsius of CO2gas incubator Digestion, brighten when observing that attached cell is gradually rounded under the microscope and progressively disengage culture dish and float in cell pancreatin When, the second attached cell culture medium described in 1mL is added into culture dish, is mixed, to obtain cell suspension；(8) cell suspension is transferred to by carrying out the generation of Secondary Culture 2~20 in the coated culture device of gelatin, to obtain Obtain the donorcells of body-cell neucleus transplanting.
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| CN111088227A (en) * | 2019-12-31 | 2020-05-01 | 广州航华生物医药科技有限公司 | Cell separation culture solution and T cell separation culture method |
| CN111004780B (en) * | 2019-12-31 | 2022-05-13 | 广州航华生物医药科技有限公司 | Purification and separation culture method for improving killing activity of immune cells |
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