CN104513807A - Method for separating cells from blood and cultivating the cells and method for cloning non-human animal - Google Patents
Method for separating cells from blood and cultivating the cells and method for cloning non-human animal Download PDFInfo
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Abstract
The invention provides a method for separating cells from blood and cultivating the cells and a method for cloning non-human animals. The method for separating cells from blood and cultivating the cells comprises following steps: (1) adding the blood to an upper layer of a separation liquid and performing centrifugation in a manner of density gradient centrifugation for obtaining mononuclear cells; (2) inoculating the mononuclear cells onto an in-vitro cultivation device containing an adherent cell culture medium to obtain adherent cells; and (3) performing serial subcultivation to the adherent cells to obtain somatic cell nuclear transplanted donor cells. The somatic cell nuclear transplanted donor cells obtained through the method can be directly used for manually cloning the non-human animals. The method is simple in operation processes, is easy to carry out, is low in cost, is less in stress stimulation and harm in animals, and can ensure a complete appearance of the animals and improve animal welfare.
Description
Technical field
The present invention relates to biology field, particularly, relate to the method for separation, culturing cell from blood and the method for clone non-human animal.
Background technology
Body-cell neucleus transplanting, also known as clone, is the most frequently used technique means of System in Animal Cell Biotechnology.By electric shocking method or direct microinjection, somatocyte higher for differentiation degree is transferred in ovocyte, then through chemokinesis, makes its reprogramming develop into embryo, transplanted back in foster mothers, birth complete individuals.Traditional somatic cell nuclear transfer technique needs costliness, accurate micromanipulation system, very high to the technical requirements of operator, is not suitable for large-scale cloned animal and produces.
Calendar year 2001, Danish scientist Gabor Vajta proposes handmade cloning technology first, the maximum key point of this technology utilizes blade to realize " cutting stoning ", thus be completely free of micromanipulation system, the instrument reducing this technology of clone drops into and artificial expenditure cost, and making this technology of clone be applied to following industrialization becomes possibility.The blastocyst rate of current body-cell neucleus transplanting is between 20%-30%, and handmade cloning animal blastocyst rate can be increased to 40%-60%.
Thus, still have much room for improvement for the donor cell source of somatic cell cloning at present.
Summary of the invention
The present invention is intended to one of solve the problems of the technologies described above at least to a certain extent.
The present invention be contriver based on following fact-finding: in recent years, cloned animal more and more has its wide application prospect, such as can be used for high-quality poultry kind cultivation, to be conducive to animals on the brink of extinction Germ-plasma resources protection; Produce all kinds of laboratory animal, obtain laboratory animal thus and there is the feature such as genetic material homogeneity, phenotype consistence, be more suitable for the every research of pharmacology; Clone is applied to transgenosis, high-level efficiency can obtain transgenic animal, shorten experimental period, obtain transgenic animal in a large number.In the process of body-cell neucleus transplanting, donorcells is of a great variety, comprises embryonic stem cell, the fat mesenchymal stem cell with multipotency, and terminal differentiation cell inoblast, liver cell, granulosa cell, liver cell etc.Derived from embryonic stem cells is in the inner cell mass of blastula stage, feeder layer cells is cultivated and can exceed generation up to a hundred, but complex operation, being unsuitable for building on a large scale is go down to posterity, and due to the singularity in its source, cannot obtain the relevant information of adult animal, for high-quality germline, clone has certain limitation; And the mescenchymal stem cell attached cell in bone source, mainly obtain target cell by the marrow in separating animal's femur, but the sample adopting this method to collect, need animal first to be put to death to facilitate gather; Other source for donor cell, all can cause tissue, organ wounds to animal individual in varying degrees.Current one-tenth fiber is the clone body cell type commonly used the most, but for large animals such as pig, ox, sheep, substantially be all gather its ear-edge tissue to carry out original cuiture in vitro, but due to the reason such as growing environment factor of itself, sample collecting be difficult to ensure aseptic, the later stage cultivate in vitro bacterium in preparation process, fungal contamination probability very high.
For this reason, one object of the present invention is that a kind of stress stimulation to animal of proposition is few, it is little to injure, and what guarantee animal appearance was complete is separated from blood, the method for culturing cell.The method comprises the following steps: utilize the mode of density gradient centrifugation to carry out after described blood is joined parting liquid upper strata centrifugal, to obtain mononuclearcell; Described mononuclearcell is inoculated in the vitro culture equipment containing attached cell substratum, to obtain attached cell; And described attached cell is carried out Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting.By according to the donorcells of the extracting from blood of the embodiment of the present invention, body-cell neucleus transplanting prepared by the method for culturing cell for handmade cloning body-cell neucleus transplanting, blastaea efficiency is reaching as high as 55%, and average blastocyst rate is about 40%.Thus, according to being separated from blood of the embodiment of the present invention, the method for culturing cell not only operating process simple, be easy to realize, cost is lower, and the method stimulates less Animal stress, injures little, can ensure the integral outer appearance of animal, improve animal welfare.
In addition, following additional technical characteristic can also be had according to the method for separation, culturing cell from blood of the embodiment of the present invention:
According to embodiments of the invention, described blood is from peripheral blood.Because peripheral blood acquisition operations is relatively simple, stimulates less, injure little to Animal stress, not only can improve animal welfare thus, and blood and the external world are comparatively closed, later stage pollution of cell culture rate is zero substantially, improves cell survival rate.
According to embodiments of the invention, described parting liquid is neutrophil leucocyte parting liquid.Thus, can effectively by utilizing the mode of density gradient centrifugation the various cellular constituents in blood to be separated according to the size of density, be divided into some layers through centrifugally operated blood, containing different blood cell components in every layer, thus effectively obtain mononuclearcell.
According to embodiments of the invention, the density of described neutrophil leucocyte parting liquid is 1.077g/mL, and wherein, described neutrophil leucocyte parting liquid contains 57g/L ficoll and 90g/L Sodium Diatrizoate.Thus, the neutrophil leucocyte parting liquid containing mentioned component improves the separation efficiency of different cellular constituent in separating blood, thus effectively can obtain the mononuclearcell blood from wherein mononuclear cell layer.
According to embodiments of the invention, described mononuclearcell is collected from the centrifugal rear middle layer of described parting liquid.After utilizing the mode of density gradient centrifugation to be added by blood to carry out centrifugation in above-mentioned parting liquid, blood is divided into some layers in above-mentioned parting liquid, respectively containing different cellular constituent in every layer, thus, directly can collect mononuclearcell from middle layer, not only experimental implementation process simple, be easy to experiment, and cellular constituent in after layering every layer is single, which thereby enhances the separation efficiency of mononuclearcell.
According to embodiments of the invention, described mononuclearcell is inoculated in the vitro culture equipment containing attached cell substratum, to obtain attached cell to comprise further: first, utilize the first attached cell substratum to cultivate 48 hours in the CO2gas incubator of 37 degrees Celsius by with the mononuclearcell after PBS cleaning, described first attached cell cultivates bFGF and the 30IU/mL heparin comprising low sugar-MEM, 15%FBS, 1 × non-essential amino acid, 10ng/mL; Then, substratum is replaced by the second attached cell substratum and continues to cultivate in the CO2gas incubator of 37 degrees Celsius, described second attached cell substratum comprises the bFGF of MEM-α, 15%FBS, 1 × non-essential amino acid and 10ng/mL.Thus, first mononuclearcell through being separated can carry out preliminary adaptability vitro culture under suitable nutritional condition, this mononuclearcell is through the first attached cell culture medium culturing containing low sugar-MEM after 48 hours, the the second attached cell substratum be replaced by by substratum containing MEM-α continues to cultivate, thus the good cell of adherent property can be obtained, and grow up in spindle shape under microscope, its nucleus is larger, growth is very fast, is therefore suitable for the experiment of handmade cloning body-cell neucleus transplanting.
According to embodiments of the invention, described vitro culture equipment is the culture dish of gelatin bag quilt or the culture plate of gelatin bag quilt.Due to, gelatin is the major structural components of the extracellular matrix of multicellular organism, can promote the adherent of cell and propagation, specific morphological and functional expression, be usually used in the vitro culture of the primary cell of various cell.Not only can improve the adherent efficiency of mononuclearcell in culture device thus, and urge adherent class Reagent evaluation ratio with other, experimental cost is lower.
According to embodiments of the invention, described attached cell is carried out Secondary Culture and comprises further, by described attached cell Secondary Culture 2-20 generation.Thus, can by being in logarithmic phase, the mononuclearcell that active good, nucleus is larger is further used for the experiment of handmade cloning body-cell neucleus transplanting.
Another object of the present invention is to provide a kind of method of cloning non-human animal, comprising: the method utilizing above-mentioned separation, culturing cell from blood, prepare the donorcells of somatic cell cloning; And based on the donorcells of described body-cell neucleus transplanting, by handmade cloning, carry out clone non-human animal.Thus, can utilize be separated from blood according to the embodiment of the present invention above-mentioned, cultured cells, by handmade cloning, obtain non-human animal.Due to by according to being separated from blood of the embodiment of the present invention, the method for culturing cell not only experimental implementation process simple, be easy to realize, cost is lower, and the method to Animal stress stimulate less, injure little, the integral outer appearance of animal can be ensured, improve animal welfare, therefore the donorcells of the body-cell neucleus transplanting of acquisition is used for non-human animal clone and have broad application prospects.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 is according to being separated from blood of the embodiment of the present invention, the method schematic diagram of culturing cell.
Fig. 2 is the method schematic diagram carrying out non-human cell clone according to the cell of utilization separation and Culture from blood of the embodiment of the present invention.
The cytological map that Fig. 3 is observed by 200 times of phase microscopes after cultivating 7d after showing the pig precaval vein density gradient separation according to the embodiment of the present invention.
The cytological map that Fig. 4 is observed by 200 times of phase microscopes after cultivating 7d after showing the pig precaval vein density gradient separation according to the embodiment of the present invention, wherein part cell is contact inhibition growth.
Fig. 5 is shown and carries out adherent culture according to the isolated cell from blood of the embodiment of the present invention and be passaged to the P1 cytological map observed by 100 times of phase microscopes of generation.
Fig. 6 is shown and carries out adherent culture according to the isolated cell from blood of the embodiment of the present invention and be passaged to the P1 cytological map observed by 200 times of phase microscopes of generation.
Fig. 7: show and separation, cultured cells are used for body-cell neucleus transplanting, the blastaea aspect graph arrived by 100 times of Huffman microscopic examinations for the 5th day according to the embodiment of the present invention.
Fig. 8: show the flow cytometric analysis results figure according to the embodiment of the present invention:
8A is the fluidic cell result figure of the ovocyte of surface markers CD29;
8B is the fluidic cell result figure of the ovocyte of surface markers CD90;
8C is the fluidic cell result figure of the ovocyte of surface markers CD34;
8D is the fluidic cell result figure of the ovocyte of surface markers CD45.
Fig. 9 is according to the method for the embodiment of the present invention donorcells based on the body-cell neucleus transplanting of the embodiment of the present invention, and by non-human animal that handmade cloning obtains.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Be exemplary below by the embodiment be described with reference to the drawings, be intended to for explaining the present invention, and can not limitation of the present invention be interpreted as.
In addition, term " first ", " second " only for describing object, and can not be interpreted as instruction or hint relative importance or imply the quantity indicating indicated technical characteristic.Thus, be limited with " first ", the feature of " second " can express or impliedly comprise one or more these features.In describing the invention, the implication of " multiple " is two or more, unless otherwise expressly limited specifically.
The present invention be contriver based on following fact-finding: in recent years, cloned animal more and more has its wide application prospect, such as can be used for high-quality poultry kind cultivation, to be conducive to animals on the brink of extinction Germ-plasma resources protection; Produce all kinds of laboratory animal, obtain laboratory animal thus and there is the feature such as genetic material homogeneity, phenotype consistence, be more suitable for the every research of pharmacology; Clone is applied to transgenosis, high-level efficiency can obtain transgenic animal, shorten experimental period, obtain transgenic animal in a large number.In the process of body-cell neucleus transplanting, donorcells is of a great variety, comprises embryonic stem cell, the fat mesenchymal stem cell with multipotency, and terminal differentiation cell inoblast, liver cell, granulosa cell, liver cell etc.Derived from embryonic stem cells is in the inner cell mass of blastula stage, feeder layer cells is cultivated and can exceed generation up to a hundred, but complex operation, being unsuitable for building on a large scale is go down to posterity, and due to the singularity in its source, cannot obtain the relevant information of adult animal, for high-quality germline, clone has certain limitation; And the mescenchymal stem cell attached cell in bone source, mainly obtain target cell by the marrow in separating animal's femur, but the sample adopting this method to collect, need animal first to be put to death to facilitate gather; Other source for donor cell, all can cause tissue, organ wounds to animal individual in varying degrees.Current one-tenth fiber is the clone body cell type commonly used the most, but for large animals such as pig, ox, sheep, substantially be all gather its ear-edge tissue to carry out original cuiture in vitro, but due to the reason such as growing environment factor of itself, sample collecting be difficult to ensure aseptic, the later stage cultivate in vitro bacterium in preparation process, fungal contamination probability very high.
Thus in first of the present invention, the present invention proposes a kind of method of separation, culturing cell from blood.The method comprises the following steps: utilize the mode of density gradient centrifugation to carry out after described blood is joined parting liquid upper strata centrifugal, to obtain mononuclearcell; Described mononuclearcell is inoculated in the vitro culture equipment containing attached cell substratum, to obtain attached cell; And described attached cell is carried out Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting.By according to the donorcells of the extracting from blood of the embodiment of the present invention, body-cell neucleus transplanting prepared by the method for culturing cell for handmade cloning body-cell neucleus transplanting, blastaea efficiency is reaching as high as 55%, and average blastocyst rate is about 40%.Thus, according to being separated from blood of the embodiment of the present invention, the method for culturing cell not only operating process simple, be easy to realize, cost is lower, and the method stimulates less Animal stress, injures little, can ensure the integral outer appearance of animal, improve animal welfare.
Below in conjunction with accompanying drawing 1 and embodiment to being of the present inventionly separated from blood, the method for culturing cell is described in further details:
S101: utilize the mode of density gradient centrifugation to carry out after blood being joined parting liquid upper strata centrifugal, to obtain mononuclearcell
According to embodiments of the invention, in separating blood cell mode and be not particularly limited, as long as the object that the mode that can reach adopted blood collection stimulates injury little to animal, the blood collection method that this area is conventional can be adopted, according to some embodiments of the invention, blood collection can be carried out by modes such as tail vein, auricular veins, according to a concrete example of the present invention, peripheral blood can be extracted by using the syringe of 50mL directly to extract pig precaval vein blood mode with it from pig.Because peripheral blood acquisition operations is relatively simple, stimulates less, injure little to Animal stress, not only can improve animal welfare thus, and blood and the external world are comparatively closed, later stage pollution of cell culture rate is zero substantially, improves cell survival rate.
Embodiments of the invention, the mode of preserve the peripheral blood of above-mentioned acquisition being also not particularly limited, as long as can prevent gather obtain peripheral blood solidify before carrying out separating step, the anti-freezing mode that this area is conventional can be adopted, according to a particular embodiment of the invention, can adopt and in gathered peripheral blood, add heparin carry out anti-freezing.The discovery that contriver is surprised, adds 125IU heparin and can effectively prevent gathered peripheral blood from solidifying in the peripheral blood that 10mL gathers.In addition, according to embodiments of the invention, preservation transport under the peripheral blood adding heparin is placed in 4 degrees celsius, is carried out.Thus, not only can prevent gathered peripheral blood from solidifying, and can prevent the effective active cellular constituent in peripheral blood from degrading, thus be beneficial to next step isolated cell operation.
Owing to containing the various cells such as red corpuscle, eosinophilic granulocyte, basophilic granulocyte, neutrophil leucocyte, T lymphocyte, B lymphocyte, natural killer cell, scavenger cell, monocyte, reticulocyte, plasmocyte, mastocyte, thrombocyte, mesenchymal cell, inoblast, adipocyte and skein cell in blood, therefore above-mentioned cell is needed to be separated, from the donorcells use wherein extracting body-cell neucleus transplanting, thus for handmade cloning.According to embodiments of the invention, from blood, be separated the mode of mononuclearcell and be not particularly limited, the various cellular segregation modes that this area is conventional can be adopted, according to concrete example of the present invention, can utilize and above-mentioned gathered peripheral blood is added parting liquid upper strata with the method for density gradient centrifugation, above-mentioned peripheral blood is separated.Thus, parting liquid can be effectively utilized with the mode of density gradient centrifugation, the various cellular constituents in blood are separated according to the size of density, be divided into some layers through centrifugally operated blood, containing different blood cell components in every layer, thus effectively obtain mononuclearcell.
According to embodiments of the invention, the parting liquid be separated above-mentioned peripheral blood adopted also is not particularly limited, according to concrete example of the present invention, described parting liquid is neutrophil leucocyte parting liquid, the density of described neutrophil leucocyte parting liquid is 1.077g/mL, and the concrete composition of neutrophil leucocyte parting liquid described in tool is 57g/L ficoll and 90g/L Sodium Diatrizoate.Thus, the neutrophil leucocyte parting liquid containing mentioned component improves the separation efficiency of different cellular constituent in separating blood, thus effectively can obtain the mononuclearcell blood from the above-mentioned mononuclear cell layer through layering peripheral blood.According to embodiments of the invention, blood ingredient after centrifugal is divided into some layers according to different density, in test tube, layering from top to bottom includes but not limited to plasma layer, cellular layer, separation liquid layer, neutrophil leucocyte layer, remaining is separated liquid layer and erythroprecipitin layer, and wherein said mononuclearcell mediates Yunfu shape cellular layer.According to embodiments of the invention, collect the method for the mononuclearcell be separated and be not particularly limited, the various collection methods can commonly used for this area, according to concrete example of the present invention, whole pipet can be adopted to draw out by the mononuclearcell in the shape cellular layer of middle Yunfu, and further the mononuclearcell drawn out is cleaned, according to a particular embodiment of the invention, the number of times of cleaning can be 2-3 time.Thus, the separation efficiency to blood cell can be improved, thus obtain high-quality mononuclearcell.
S102: mononuclearcell is inoculated in vitro culture equipment, to obtain attached cell
According to embodiments of the invention, to isolate above-mentioned and the mononuclearcell cleaned is inoculated in vitro culture equipment containing attached cell substratum, to obtain attached cell.According to embodiments of the invention, the mode of vitro culture is carried out to isolated mononuclearcell and is not particularly limited, as long as the adherent normal growth of above-mentioned mononuclearcell can be made, the various extracorporeal culturing methods that this area is conventional can be adopted, according to some embodiments of the present invention, first the first attached cell substratum is utilized to cultivate 48 hours in the CO2gas incubator of 37 degrees Celsius the mononuclearcell through cleaning.According to embodiments of the invention, the composition of described first attached cell substratum is not particularly limited, as long as be applicable to just carrying out adherent growth from blood separation mononuclearcell out in vitro culture device containing enough nutrition, the various adherent culture bases that this area is conventional can be adopted, according to a particular embodiment of the invention, described first attached cell substratum can comprise bFGF and the 30IU/mL heparin of low sugar-MEM, 15%FBS, [1 ×] non-essential amino acid, 10ng/mL.According to some embodiments of the present invention, after 48 hours, described first attached cell substratum is replaced by the second attached cell substratum to continue to cultivate in the CO2gas incubator of 37 degrees Celsius, described second attached cell substratum volume can comprise the bFGF of MEM-α, 15%FBS, [1 ×] non-essential amino acid and 10ng/mL.Wherein, according to embodiments of the invention, after 48 hours, described first attached cell substratum is replaced by the mode of the second attached cell substratum and is not particularly limited, according to concrete example of the present invention, first described first attached cell substratum can be discarded, then PBS is adopted to clean to remove the first residual attached cell substratum to cell, again the second attached cell substratum is joined in adherent mononuclearcell and proceed to cultivate, according to embodiments of the invention, the number of times of cleaning can be 1-2 time.Wherein, MEM-α is a kind of MEM substratum of improvement, with the addition of multiple non-essential amino acid and vitamin B12, C etc.; And use low sugar-MEM can promote cell attachment, use MEM-α after 48h instead and can reduce adherent hemocyte, thus be conducive to adherent karyocyte growth.Thus, first mononuclearcell through being separated can carry out preliminary adaptability vitro culture (i.e. the first attached cell culture medium culturing of utilization containing low sugar-MEM 48 hours) under suitable nutritional condition, substratum is replaced by the second attached cell substratum containing MEM-α being more suitable for adherent growth to continue to cultivate, thus makes the above-mentioned mononuclearcell separated from peripheral blood to form suitable in nutritive ingredient and to carry out suitable growth in the adherent culture base of abundance.
According to embodiments of the invention, the vitro culture equipment adopted also is not particularly limited, as long as be applicable to isolated mononuclearcell growth from blood, the conventional various culture device in this area can be adopted and select as required, such as 35mm culture dish, 60mm culture dish, 100mm culture dish, 6 orifice plates and 4 orifice plates, according to concrete example of the present invention, the vitro culture adopted can be the culture dish of gelatin bag quilt or the culture plate of gelatin bag quilt.Due to, gelatin is the major structural components of the extracellular matrix of multicellular organism, can promote the adherent of cell and propagation, specific morphological and functional expression, be usually used in the vitro culture of the primary cell of various cell.Not only can improve the adherent efficiency of mononuclearcell in culture device thus, and reduce experimental cost.According to embodiments of the invention, adopt the and mode that attached cell substratum is cultivated adherent mononuclearcell being not particularly limited, according to concrete example of the present invention, can adopt and within 2-3 days, once change liquid operation thus adherent mononuclearcell can be kept to be in nutritive ingredient be all the time applicable to and grow in the attached cell substratum of abundance.Good according to the adherent property of mononuclearcell that the method for the embodiment of the present invention obtains, growth is very fast, grows up under microscope in spindle shape, and its entoblast is comparatively large and high-visible, is therefore suitable for handmade cloning body-cell neucleus transplanting (see figure 3).
S103: attached cell is carried out Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting
According to embodiments of the invention, the (see figure 4) when adherent mononuclearcell becomes the growth of bulk contact inhibition dress, can carry out passage cultivation.According to concrete example of the present invention, can by described attached cell Secondary Culture 2-20 generation.According to embodiments of the invention, the adherent mononuclearcell of the above-mentioned dress growth in bulk contact inhibition is carried out the mode of Secondary Culture and is not particularly limited, the various propagating methods can commonly used for this area, according to concrete example of the present invention, following steps can be adopted: discard former substratum; Add PBS damping fluid rinse cell 2 times; After discarding PBS damping fluid, cell dissociation buffer is added cell, and cell is positioned in the CO2gas incubator of 37 degrees Celsius and digests; Add adherent culture base 2 and mix, to obtain cell suspension with the mononuclearcell through digesting; Above-mentioned cell suspension is transferred in the culture device of gelatin bag quilt, carries out Secondary Culture.Mononuclearcell growth is obtained vigorous, proliferative good (see Fig. 5 and Fig. 6) by the method for the cultured cell line of the embodiment of the present invention.Wherein, according to embodiments of the invention, the Digestive system adopted also is not particularly limited, as long as examine under a microscope can make attached cell become gradually circle brighten, and depart from cell cultivation equipment gradually thus float in cell dissociation buffer, can adopt the various cell dissociation buffers that this area is conventional, according to a particular embodiment of the invention, cell dissociation buffer can be 0.05% pancreatin-200mg/L EDTA.Thus, can by being in logarithmic phase, the mononuclearcell that active good, nucleus is larger 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 that non-human animal is cloned, comprising: the method utilizing above-mentioned separation, culturing cell from blood, prepare the donorcells of somatic cell cloning; And based on the donorcells of described body-cell neucleus transplanting, by handmade cloning, carry out clone non-human animal.Thus, can utilize be separated from blood according to the embodiment of the present invention above-mentioned, cultured cells, by handmade cloning, obtain non-human animal.Due to by according to being separated from blood of the embodiment of the present invention, the method for culturing cell not only experimental implementation process simple, be easy to realize, cost is lower, and the method to Animal stress stimulate less, injure little, the integral outer appearance of animal can be ensured, improve animal welfare, therefore the donorcells of the body-cell neucleus transplanting of acquisition is used for non-human animal clone and have broad application prospects.
The method that cell to utilization of the present invention separation and Culture from blood carries out non-human cell clone below in conjunction with accompanying drawing 2 and embodiment is described in further details:
S201: oocyte IVM and surface molecular qualification
According to embodiments of the invention, the mode of oocyte collection is also not particularly limited, as long as healthy ovum can be obtained, the various methods that this area is conventional can be adopted, according to a particular embodiment of the invention, can comprise the following steps: the ovary of collection to be placed in the container that physiological saline is housed and to keep 35 degrees Celsius of constant temperature; Then above-mentioned ovary is positioned in the culture dish that embryo's washings is housed, utilizes blade to be scratched by ovarian follicle, to make ovum flow out; And above-mentioned ovum is collected in pre-balance be cultured to egg maturation containing in the culture device of ovum substratum.According to embodiments of the invention, ovum is cultured to ripe condition to be not particularly limited, the various ovum culture techniques can commonly used for this area, according to concrete example of the present invention, in order to reach the metastable culture environment of pH value, the CO2gas incubator in advance substratum being placed on 5% concentration balances, then ovum is collected in pre-balance containing in embryo maturation substratum (IVM) four orifice plate, 50, each hole embryo, and the ripe 42h of the CO2gas incubator above-mentioned four hole versions being loaded with ovum being placed in 38.5 degrees Celsius.According to embodiments of the invention, above-mentioned embryo's washings is also not particularly limited, can be the various liquid that embryo is washed that this area is commonly used, according to concrete example of the present invention, comprise in the embryo's washings (ASP) adopted: the Sodium.alpha.-ketopropionate of the 10 × TCM199 of 10%v/v, the sodium bicarbonate of 0.17mg/mL, 0.20mg/mL, 3.60mg/mL He Peisi sodium salt, the He Peisi acid of 2.63mg/mL, the glutamine of 0.20mg/mL, the amphotericin B of 3.00 μ g/mL, the heparin of 30IU/mL, the serum of 2%.Thus, the fragment in ovary cut substrate can be removed, thus reduce institute and cultivate the pollution probability of object cell, raising cell survival rate.
According to embodiments of the invention, embryo maturation substratum (IVM) is also not particularly limited, the substratum that embryo is cultivated can commonly used for this area, according to specific examples of the present invention, the embryo maturation substratum (IVM) adopted comprises: the glutamine of the human chorionic gonadotrophin of TCM199, the 5IU/mL of [1 ×], the pregnant mare serum of 1IU/mL, 0.10mg/mL, 0.05mg/mL gentamicin, 10% liquor folliculi, 10%v/v serum.Thus, by making ovum in this substratum through the cultivation of 42 hours, the maturation of ovocyte can be promoted, discharging first polar body, thus improving into heat efficiency (first polar body rate of discharge), being about greater than 80%.
According to embodiments of the invention, be not particularly limited the method that obtained ovocyte is identified, the various cellular identification methods can commonly used for this area, include but not limited to cell surface molecule labelling method, flow cytometry etc.According to a particular embodiment of the invention, CD29, CD90, CD34, CD45, FITC Mouse IgG1 antibody different with PE Mouse IgG17 kind is utilized to carry out surface markers to obtained ovocyte, then BD FACSCalibur flow cytometer and WinMDI2.8 software is utilized to analyze, to determine whether obtained cell is mescenchymal stem cell.
S202: ovocyte takes off ovarian cumulus and stoning
The ovum of above-mentioned cultivation maturation is transferred in Unidasa, to slough cumulus granulosa cells from four orifice plates containing embryo maturation substratum (IVM).According to embodiments of the invention, from four orifice plates containing embryo maturation substratum (IVM), be transferred to the jump operation in Unidasa by cultivating ripe ovum and be not particularly limited, it can be the technology of the transfer ovum that this area is commonly used, according to a particular embodiment of the invention, utilize liquid-transfering gun by maturation completely ovocyte from four orifice plates containing embryo maturation substratum (IVM) in Unidasa.Wherein, according to embodiments of the invention, the concentration of the Unidasa adopted also is not particularly limited, as long as can cumulus granulosa cells be sloughed, those skilled in the art can carry out suitable adjustment according to the kind of the concentration of cell and reagent, according to a particular embodiment of the invention, the concentration of the Unidasa adopted can be 1mg/mL.Unidasa Main Function dissolves hyaluronic acid between cumulus cell, thus cumulus cell is disperseed, and thus, can adopt suitable concentration by the cumulus cell dispersion in the ovum of above-mentioned cultivation maturation to remove further.According to embodiments of the invention, slough the operation of cumulus granulosa cells and be not particularly limited, the various technology that this area is conventional can be adopted, according to specific embodiment of the invention profit, the discovery that contriver is surprised, adopt the liquid-transfering gun with rifle head to carry out resorption and beat Unidasa containing ovocyte, what can utilize rifle head small-borely repeatedly carries out suction to ovum and beats, thus sloughs the cumulus granulosa cells that mature egg pericellular carries out with it metabolism contact.
According to embodiments of the invention, remove the method for ootid nucleus and be not particularly limited, the various pitting methods can commonly used for this area, according to specific examples of the present invention, first the above-mentioned exposed ovocyte eliminating cumulus cell can be positioned in proteolytic enzyme, so as by the zona pellucida on ovocyte top layer digestion loose, then transfer them to second embryo operation liquid operation drip in, and according to the method that polar body is located, first polar body is excised together with part ovocyte matter.According to embodiments of the invention, the second embryo operation liquid can comprise the sodium bicarbonate of [10 ×] TCM199,0.17mg/mL of 10%v/v, the Sodium.alpha.-ketopropionate of 0.20 mg/mL, the He Peisi sodium salt of 3.60mg/mL, the He Peisi acid of 2.63mg/mL, the glutamine of 0.20mg/mL, 2.5 μ g/mL B cytochalasin Bs and 20% serum.Thus, effectively oocyte nuclei can be removed.
According to embodiments of the invention, place the above-mentioned mode through non-nucleus egg mother cell and be not particularly limited, those skilled in the art can need to select according to subsequent operations, according to concrete example of the present invention, above-mentioned non-nucleus egg mother cell is transferred to first embryo operation liquid operation drip in stand-by.According to embodiments of the invention, the first embryo operation liquid can comprise the sodium bicarbonate of [10 ×] TCM199,0.17mg/mL of 10%v/v, the Sodium.alpha.-ketopropionate of 0.20mg/mL, the He Peisi sodium salt of 3.60mg/mL, the He Peisi acid of 2.63mg/mL, the glutamine of 0.20mg/mL, the serum of 2%.Thus, what obtain survives in containing optimal embryo operation pendular ring border through non-nucleus egg mother cell.
S203: ovocyte and donorcells merge
According to embodiments of the invention, be first scattered in by the somatocyte obtained in S103 during above-mentioned first embryo operation liquid operation drips, thus, somatocyte divides cloth-like in individual cells in the operation of this first embryo operation liquid is dripped; Then place to transfer to after for some time in phytohemagglutinin through non-nucleus egg mother cell above-mentionedly to drip what obtain in S202 containing somatic first embryo operation liquid operation, wherein according to specific exampless more of the present invention, by placing 1 ~ 2 second in 1mg/mL phytohemagglutinin through non-nucleus egg mother cell of obtaining in S202, cytosolic surface band toughness can be made thus, thus make single somatocyte and fit fully in the first embryo operation liquid operation is dripped through non-nucleus egg mother cell.
According to embodiments of the invention, non-nucleus egg mother cell (kytoplasm) and donorcells carried out the method that merges and be not particularly limited, can be the various cell-fusion techniques that this area is commonly used, according to concrete example of the present invention, can kytoplasm-somatocyte be transferred in the integration slot being loaded with mixing operation liquid, be merged by electric shock.According to embodiments of the invention, the mixing operation liquid in integration slot can comprise 3M N.F,USP MANNITOL, 1mg/mL polyvinyl alcohol and 0.1mM magnesium sulfate.Ovocyte and somatocyte can be in optimal envrionment conditions and accept electric shock thus, thus improve fusion efficiencies.According to embodiments of the invention, the condition merge ovocyte-somatocyte is also not particularly limited, those skilled in the art can regulate according to concrete cell situation and equipment situation, according to embodiments of the invention, electric shock ovocyte and somatocyte carry out the condition that merges and can to shock by electricity 9 μ s for 100V direct current, 4V alternating-current electric shock.By using alternating-current can make kytoplasm depolarize, thus it can be made to hang on wire electrode, and by electric pulse, cytolemma can be made to form an instantaneous perforation, thus make kytoplasm be combined with donorcells film and merge.Thus, kytoplasm and somatocyte can accept electric shock under optimal condition, thus are merged fully, to obtain reconstruct clone embryos.According to embodiments of the invention, the cell shocked by electricity is transferred to embryo operation liquid 1 operate drip in, and operation board is placed in 38.5 degrees Celsius of thermal station wait for 1 hour, thus improve late embryogenesis grow efficiency.
S204: activate reconstructed embryo, forms embryo
According to embodiments of the invention, by the integration slot that has somatic kytoplasm to be transferred to by melting of obtaining in S203 step to be loaded with activation manipulation liquid, and hang the female kytoplasm of the stoning ovum obtained in S202 step, then shock by electricity.Wherein utilize alternating-current to make reconstructed embryo depolarize, thus make it can hang on wire electrode, second kytoplasm is also by unpolarizing, and it can be made can to contact with reconstructed embryo.By aforesaid method, make an individual cells merge two kytoplasms, and the composition of kytoplasm increase, and can provide more nutritive substance, be conducive to the fetal development in later stage.According to embodiments of the invention, above-mentioned activation manipulation liquid can be the N.F,USP MANNITOL of 3M, 1mg/mL polyvinyl alcohol, the magnesium sulfate of 0.1mM and the calcium chloride of 0.05mM.
According to embodiments of the invention, carry out the condition that shocks by electricity and be not particularly limited, those skilled in the art can regulate, according to specific examples of the present invention according to concrete cell situation and equipment situation, the condition of electric shock can to shock by electricity 80 μ s for 43V direct current, then adopts 4V alternating-current to shock by electricity.Thus, by using alternating-current can make reconstructed embryo depolarize, thus it can be made to hang on wire electrode, and by electric pulse, cytolemma can be made to form an instantaneous perforation, thus make reconstructed embryo be combined with cytoplasmic cell membrane and merge; Then by electric shock after embryo be transferred to first embryo operation liquid operation drip in, treat two cytomixis.Thus, the stability of embryo can be improved.
According to embodiments of the invention, the mode activated the above-mentioned cell passing through fusion is unrestricted, the various activation techniques can commonly used for this area, according to a particular embodiment of the invention, embryo through merging is carried out chemokinesis, wherein according to specific examples of the present invention, the embryo through merging is transferred to during chemokinesis drips and carries out chemokinesis.According to some embodiments of the present invention, above-mentionedly carry out chemically sensitized chemokinesis and drip B cytochalasin B containing embryo culture medium (IVC) and 5 μ g/mL, the cycloheximide of 10 μ g/mL.According to embodiments of the invention, embryo culture medium (IVC) can comprise the sodium-chlor of 6.31mg/mL, 2.11mg/mL sodium bicarbonate, the Repone K of 0.75mg/mL, the potassium primary phosphate of 0.05mg/mL, the magnesium sulfate of 0.05mg/mL, the calcium lactate of 0.62mg/mL, the Sodium.alpha.-ketopropionate of 0.02mg/mL, the meso-inositol of 0.50mg/mL, 0.01g/mL's is phenol red, the glutamine of 0.05mg/mL, the sub-ox acid iodide of 0.55mg/mL, the gentamicin of 0.04mg/mL, the bovine serum albumin of 3g/L, the non-essential amino acid of [1 ×] and the indispensable amino acid of [0.5 ×].The above-mentioned cell through merging can be made thus to be activated in optimal medium component.According to other embodiments of the present invention, the embryo through merging is transferred in embryo culture medium carry out chemokinesis 38.5 degrees Celsius, carry out in 5% carbonic acid gas and 5% oxygen incubator.According to concrete example of the present invention, the embryo through merging is transferred in embryo culture medium and 38.5 degrees Celsius, cultivate 4 hours in 5% carbonic acid gas and 5% oxygen incubator.Thus, the above-mentioned cell through activating can be made under the envrionment conditions of the most applicable growth to carry out adaptability growth, thus improve survival rate of embryo.
According to embodiments of the invention, by above-mentioned through to activate and the embryo of adaptability growth is transferred to containing in four orifice plates of embryo culture medium of punching from above-mentioned chemokinesis drips, and 38.5 degrees Celsius, proceed to cultivate 120h or 144h in 5% carbonic acid gas and 5% oxygen incubator.Multiple lensless Fourier transform holography reconstructed embryo can be made can to cultivate in same fluid apertures by punching, each oneself independently developmenting space own, and in same fluid apertures, message exchange closely can occur, be conducive to fetal development.Thus, the above-mentioned embryo through activation and adaptability growth can grow further under suitable conditions and environment, thus improves the surviving rate of embryo.
S205: Embryonic limb bud cell
According to embodiments of the invention, the above-mentioned Embryonic limb bud cell through vitro culture is treated pregnant animal uterine tube or intrauterine, clones non-human animal through enough pregnancy periods.According to embodiments of the invention, the above-mentioned Embryonic limb bud cell through vitro culture treated pregnant animal uterine tube or intrauterine mode and be not particularly limited, the Embryonic limb bud cell technology that usually can adopt for this area, according to a particular embodiment of the invention, the blastaea (see figure 7) of growing to 120h is entered to treat in the horn of uterus of pregnant parent by operation transplantation.According to embodiments of the invention, embryo grows about 114 days in parent, and by spontaneous labor or output of cutting open the belly clone non-human animal, birth animal sees Fig. 9.
Embodiment 1 obtains mononuclearcell from blood
In the conical centrifuge tube of 15mL, add 125IU heparin in advance, and make it be uniformly distributed in the tube wall of conical centrifuge tube by concussion.Directly extract pig precaval vein blood mode by adopting the syringe of 50mL the peripheral blood of miniature pig is about 12mL is collected in the above-mentioned conical centrifuge tube adding heparin 15mL.The conical centrifuge tube this being loaded with peripheral blood is placed in 4 degrees Celsius of ice chest transports meeting laboratories.
Separately get the conical centrifuge tube of two 15mL, and after adding the neutrophil leucocyte parting liquid (containing 57g/L ficoll and 90g/L Sodium Diatrizoate) of 5mL wherein respectively, liquid-transfering gun is utilized to press close to along the conical centrifuge tube wall of 15mL the anticoagulation cirumferential blood gathered that neutrophil leucocyte parting liquid liquid level slowly adds 5mL carefully, and ensure that this anticoagulation cirumferential blood does not mix with the neutrophil leucocyte parting liquid of lower floor, then put down centrifugal 30 minutes at 400g Water Under.
After centrifugal, utilize liquid-transfering gun to collect Yunfu, middle layer shape mononuclearcells from two centrifuge tubes respectively, and merge the conical centrifuge tube being positioned over a new 50mL.The PBS of 20mL is joined in the aforementioned conical centrifuge tube containing mononuclearcell 50mL, and utilize Pasteur's dropper repeatedly to blow and beat to clean.After cleaning, by the PBS suspension containing mononuclearcell under 300g condition centrifugal 10 minutes.After centrifugal, take out the conical centrifuge tube of 50mL, in the conical centrifuge tube of the 50mL precipitated containing mononuclearcell, after removing supernatant liquor, add the PBS of 20mL again, and again utilize Pasteur's dropper repeatedly to blow and beat to carry out second time and clean.After second time cleaning, by the PBS suspension containing mononuclearcell under 300g condition centrifugal 10 minutes again.Then repeat above-mentioned second time cleaning step, third time cleaning is carried out to mononuclearcell.
Embodiment 2 mononuclearcell vitro culture
Obtain in embodiment 1 through to clean for 3 times and discard centrifugal after PBS supernatant liquor cell precipitation in add the first attached cell substratum that 2mL contains low sugar-MEM, 15%FBS, [1 ×] non-essential amino acid, 10ng/mL bFGF and 30IU/mL heparin, and utilize Pasteur's dropper mononuclearcell to be precipitated piping and druming to be dispersed in the first attached cell substratum.By the first attached cell media transfer containing mononuclearcell in the 35mm culture dish of gelatin bag quilt, be then placed in the CO2gas incubator of 37 degrees Celsius and cultivate.
Through the cultivation of 48 hours, take out from incubator to the 35mm culture dish being loaded with mononuclearcell, after discarding the first attached cell substratum, add PBS and carry out rinsing.After discarding PBS, in the 35mm culture dish being loaded with mononuclearcell, add the second attached cell substratum containing MEM-α, 15%FBS, [1 ×] non-essential amino acid and 10ng/mL bFGF, and culture dish is put back to incubator and proceed to cultivate.And adopt the second attached cell substratum to change liquid process to the 35mm culture dish being loaded with mononuclearcell respectively the 72nd and 96 hours.This mononuclearcell was cultivated after 5-7 days, and be placed in opticmicroscope and observe, under mirror, mononuclearcell is into fibrous adherent cell growth (see figure 3) on culture dish bottom surface, and nucleus is comparatively large, and entoblast is high-visible.
The Secondary Culture of embodiment 3 mononuclearcell
The (see figure 4) when adherent mononuclearcell examines under a microscope the dress growth in bulk contact inhibition, discards the second attached cell substratum in culture dish; Then PBS damping fluid rinse cell is added 2 times; After discarding PBS damping fluid, 200 μ L0.05% pancreatin-200mg/L EDTA are added in the culture dish being loaded with adherent mononuclearcell, and cell is positioned in the CO2gas incubator of 37 degrees Celsius and digests, when examine under a microscope attached cell become gradually circle brighten and gradually depart from culture dish and float in cell pancreatin time, 1mL second attached cell substratum is added in culture dish, and by the piping and druming of Pasteur's dropper, mononuclearcell is mixed, to obtain cell suspension with the fresh second attached cell substratum added; Then above-mentioned cell suspension is transferred to and carry out Secondary Culture in the culture device of gelatin bag quilt.The adherent mononuclearcell entering Secondary Culture is examined under a microscope, and cell late growing stage is vigorous, proliferative good (see Fig. 5 and Fig. 6).The 2-20 of Secondary Culture is directly moved in 1.5mL centrifuge tube stand-by for mononuclearcell cell suspension, for handmade cloning.
Embodiment 4 oocyte IVM and surface molecular qualification
The pig ovary collected from slaughterhouse is placed on to keep osmotic balance in the container that physiological saline is housed, and transports go back to laboratory with 35 degrees Celsius of constant temperatures.Ovary to be positioned in the culture dish that embryo's washings (ASP) is housed (wherein the sodium bicarbonate of embryo's washings (ASP) 10 × TCM199 containing 10%v/v, 0.17mg/mL, the Sodium.alpha.-ketopropionate of 0.20mg/mL, 3.60mg/mL He Peisi sodium salt, the He Peisi acid of 2.63mg/mL, the glutamine of 0.20mg/mL, the amphotericin B of 3.00 μ g/mL, the heparin of 30IU/mL, the serum of 2%), with blade, ovarian follicle to be scratched the ovum in ovarian follicle is flowed out.In order to reach the metastable culture environment of pH value, the CO2gas incubator in advance substratum being placed on 5% concentration balances, then by the ovum that flows out with the density collection of every 50 embryos in hole in four orifice plates of the embryo maturation substratum (IVM) of pre-balance, wherein human chorionic gonadotrophin, the pregnant mare serum of 1IU/mL, the glutamine of 0.10mg/mL, 0.05mg/mL gentamicin, 10% liquor folliculi, the 10%v/v serum of TCM199,5IU/mL of embryo maturation substratum (IVM) containing [1 ×].Then four orifice plates being loaded with embryo are placed in 38.5 degrees Celsius of CO2gas incubator and carry out cultivation 42h to ripe.
Adherent mature oocyte is carried out digestion process, and under 350g condition centrifugal 3 minutes, after abandoning supernatant, in precipitation, add 2mL PBS carry out piping and druming and wash; And then with 350g centrifugal 3 minutes, add 700 μ L PBS to the precipitation in centrifuge tube after abandoning supernatant, obtained suspension evenly distribute joined after piping and druming mixing in 7 new 2mL centrifuge tubes, namely often pipe adds 100 μ L cell suspending liquids; Then respectively to adding the different antibody of 5 μ L ~ 20 μ L antibody 7 in each centrifuge tube: CD29, CD90, CD34, CD45, FITC Mouse IgG1 and PE Mouse IgG1, after vortex fully mixes, lucifuge hatches 15 minutes; Then carry out 2 washings to the cell that marked surface antigen, condition is and adds 1mL PBS, shake up and down even after with 350g centrifugal 3 minutes, abandon supernatant; And then dissolve the cell precipitation after washing with 0.4mL poly methyl alcohol (4% concentration is dissolved in PBS), use BD FACSCalibur to carry out upper machine analysis to it, utilize WinMDI2.8 software to read result, result is see Fig. 8.Fig. 8 shows cell high expression level CD29 according to the embodiment of the present invention and CD90 surface antigen, and does not express CD34 and CD45 surface antigen.The result of Fig. 8 shows the ovocyte high expression level CD29 and CD90 surface antigen that obtain, do not express CD34 and CD45 surface antigen, the positive rate of CD29 and CD90 of wherein obtained ovocyte is respectively 99.27% and 98.62, and the positive rate of CD34 and CD45 is respectively 0.43% and 0.45%.
Embodiment 5 porcine oocytes takes off ovarian cumulus and stoning
The ovum liquid-transfering gun of above-mentioned cultivation maturation is transferred in 1mg/mL Unidasa from four orifice plates containing embryo maturation substratum (IVM), with rifle head come resorption since and slough cumulus granulosa cells.Then first the above-mentioned exposed ovocyte eliminating cumulus cell is positioned in proteolytic enzyme, so that by loose for the zona pellucida digestion covering ovocyte top layer, then transfer them to second embryo operation liquid operation drip in, and according to the method that polar body is located, first polar body is excised together with part ovocyte matter.Non-nucleus egg mother cell is transferred to the first embryo operation liquid operation drip in stand-by, thus can surviving in containing optimal embryo operation pendular ring border through non-nucleus egg mother cell of obtaining.Wherein the first embryo operation liquid can comprise the sodium bicarbonate of [10 ×] TCM199,0.17mg/mL of 10%v/v, the Sodium.alpha.-ketopropionate of 0.20mg/mL, the He Peisi sodium salt of 3.60mg/mL, the He Peisi acid of 2.63mg/mL, the glutamine of 0.20mg/mL, the serum of 2%.
Embodiment 6 ovocyte and donorcells merge
The somatocyte that embodiment 3 obtains is placed on during the first embryo operation liquid operation drips, and makes somatocyte in this operation is dripped in individual cells point cloth-like.What embodiment 5 obtained places 1 second ~ 2 seconds through non-nucleus egg mother cell in 1mg/mL phytohemagglutinin, then the somatocyte obtained with above-described embodiment 3 merge put into aforementioned first embryo operation liquid operate drip, thus make single somatocyte and through non-nucleus egg mother cell first embryo operation liquid operation drip in fit fully.Enucleation oocyte (kytoplasm)-somatocyte is transferred in the integration slot being loaded with mixing operation liquid, is merged by electric shock, to obtain reconstruct clone embryos.Mixing operation liquid wherein in integration slot comprises 3M N.F,USP MANNITOL, 1mg/mL polyvinyl alcohol and 0.1mM magnesium sulfate.The condition that electric shock ovocyte and somatocyte carry out merging is that 100V direct current shocks by electricity 9 μ s, 4V alternating-current electric shock.Shocked by electricity all ovocytes and somatocyte successively, then transferred to first embryo operation liquid operation drip in, operation board is placed in 38.5 degrees Celsius of thermal station wait for 1 hour.
Embodiment 7 activates reconstructed embryo, forms embryo
According to embodiments of the invention, by melting in embodiment 6 in the integration slot that has somatic kytoplasm to be transferred to be loaded with activation manipulation liquid (wherein activation manipulation liquid contains the N.F,USP MANNITOL of 3M, 1mg/mL polyvinyl alcohol, the magnesium sulfate of 0.1mM and the calcium chloride of 0.05mM), and hang the stoning kytoplasm obtained in embodiment 5, then shock by electricity, to make reconstructed embryo and cytomixis: wherein utilize alternating-current to make reconstructed embryo depolarize, thus make it can hang on wire electrode, second kytoplasm is also by unpolarizing, and it can be made can to contact with reconstructed embryo.The condition of electric shock is that 43V direct current shocks by electricity 80 μ s, then adopts 4V alternating-current electric shock, and 4V alternating-current is until electric shock terminates rear closedown.Thus, by using alternating-current can make reconstructed embryo depolarize, thus it can be made to hang on wire electrode, and by electric pulse, cytolemma can be made to form an instantaneous perforation, thus make reconstructed embryo be combined with cytoplasmic cell membrane and merge; Then by electric shock after embryo be transferred to first embryo operation liquid operation drip in, treat two cytomixis.
Embryo through merging being transferred to during chemokinesis drips and carrying out chemokinesis, above-mentionedly carrying out chemically sensitized chemokinesis and drip containing embryo culture medium (IVC) and the B cytochalasin B of 5 μ g/mL and the cycloheximide of 10 μ g/mL.Wherein, embryo culture medium (IVC) comprises the sodium-chlor of 6.31mg/mL, the sodium bicarbonate of 2.11mg/mL, the Repone K of 0.75mg/mL, the potassium primary phosphate of 0.05mg/mL, the magnesium sulfate of 0.05mg/mL, the calcium lactate of 0.62mg/mL, the Sodium.alpha.-ketopropionate of 0.02mg/mL, the meso-inositol of 0.50mg/mL, the glutamine of phenol red, 0.05mg/mL of 0.01g/mL, the sub-ox acid iodide of 0.55mg/mL, the gentamicin of 0.04mg/mL, the bovine serum albumin of 3g/L, the non-essential amino acid of [1 ×] and the indispensable amino acid of [0.5 ×].Embryo through merging is transferred in embryo culture medium 38.5 degrees Celsius, carry out chemokinesis in 5% carbonic acid gas and 5% oxygen incubator, activationary time is 4 hours.
By above-mentioned through to activate and the embryo of adaptability growth is transferred to containing in four orifice plates of embryo culture medium of punching from above-mentioned chemokinesis drips, and 38.5 degrees Celsius, proceed to cultivate 120h in 5% carbonic acid gas and 5% oxygen incubator.
Embodiment 8 Embryonic limb bud cell
The above-mentioned Embryonic limb bud cell through vitro culture treats pregnant animal uterine tube or intrauterine, clones non-human animal through enough pregnancy periods.First the blastaea (see figure 7) of growing to 120h is entered to treat in the horn of uterus of pregnant sow body by operation transplantation.Embryo grows about 114 days in sow acceptor, and by spontaneous labor or output of cutting open the belly clone non-human animal, birth animal sees Fig. 9.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (9)
1. be separated from blood, the method for culturing cell, it is characterized in that, comprise the following steps:
The mode of density gradient centrifugation is utilized to carry out after described blood is joined parting liquid upper strata centrifugal, to obtain mononuclearcell;
Described mononuclearcell is inoculated in the vitro culture equipment containing attached cell substratum, to obtain attached cell; And
Described attached cell is carried out Secondary Culture, to obtain the donorcells of body-cell neucleus transplanting.
2. method according to claim 1, is characterized in that, described blood is from peripheral blood.
3. method according to claim 1, is characterized in that, described parting liquid is neutrophil leucocyte parting liquid.
4. method according to claim 3, is characterized in that, the density of described neutrophil leucocyte parting liquid is 1.077g/mL,
Wherein, described neutrophil leucocyte parting liquid contains 57g/L ficoll and 90g/L Sodium Diatrizoate.
5. method according to claim 1, is characterized in that, described mononuclearcell is collected from the centrifugal rear middle layer of described parting liquid.
6. method according to claim 1, is characterized in that, described mononuclearcell is inoculated in the vitro culture equipment containing attached cell substratum, comprises further to obtain attached cell:
First, utilize the first attached cell substratum to cultivate 48 hours in the CO2gas incubator of 37 degrees Celsius by with the mononuclearcell after PBS cleaning, described first attached cell substratum comprises bFGF and the 30IU/mL heparin of low sugar-MEM, 15%FBS, 1 × non-essential amino acid, 10ng/mL;
Then, substratum is replaced by the second attached cell substratum and continues to cultivate in the CO2gas incubator of 37 degrees Celsius, described second attached cell substratum comprises the bFGF of MEM-α, 15%FBS, 1 × non-essential amino acid and 10ng/mL.
7. method according to claim 1, is characterized in that, described vitro culture equipment is the culture dish of gelatin bag quilt or the culture plate of gelatin bag quilt.
8. method according to claim 1, is characterized in that, described attached cell is carried out Secondary Culture and comprises further, by described attached cell Secondary Culture 2-20 generation.
9. to the method that non-human animal clones, it is characterized in that, comprising:
Utilize the method according to any one of claim 1 ~ 8, prepare the donorcells of body-cell neucleus transplanting; And
Based on the donorcells of described body-cell neucleus transplanting, by handmade cloning, carry out clone non-human animal.
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| CN104513807B (en) | 2018-01-12 |
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