CN103333918A - Method for improving in-vitro development efficiency of porcine cloned embryos - Google Patents
Method for improving in-vitro development efficiency of porcine cloned embryos Download PDFInfo
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Abstract
The invention discloses a method for improving in-vitro development efficiency of porcine cloned embryos. The method comprises the following steps of: performing molecular identification on bone marrow-derived mesenchymal stem cells, and establishing a method capable of identifying the purity of the cells by using a few cells through a 6-channel flow type technology; performing gene modification on the porcine bone marrow mesenchymal stem cells to obtain porcine bone marrow mesenchymal stem cells for expressing multiple reprogramming factors; and separating out positive porcine bone marrow mesenchymal stem cells by using a fluorescent flow type separation method, continuously culturing the positive porcine bone marrow mesenchymal stem cells for 3 to 7 days, performing nuclear transfer on the cultured cells serving as nuclear donor cells, and studying the influence of 4RFs-3days, 6RFs-3days, 4RFs-7days and 6RFs-7days pMSC serving as nuclear donor cells on the improvement of the development efficiency of the porcine cloned embryos. The results show that the 4RFs-7days pMSC can well promote fission of the cloned embryos and formation of blastospheres, the cloned embryos can form a homogeneous state with uniform cell quantity, and a foundation is laid for efficiently cloning adult high-quality pig varieties in large scale.
Description
Technical field
The present invention relates to a kind of method that improves porcine clone embryos ectogenesis efficient, especially a kind of reprogrammed factor pair donorcells that utilizes carries out genetic modification, thereby improves the method for porcine clone embryos ectogenesis efficient, belongs to breeding or insemination method field.
Background technology
Pig has the very similar organ with the mankind, and similar anatomy physiological characteristic, becomes the living model material of regenerative medicine research gradually.People utilize nuclear transfer technology more and more, produce have certain hereditary defect or modifying factor pig as disease model, be used for the clinical treatment of simulating human disease better.In addition, in the actual production of live pig, the germ plasm resource of some high-qualitys is because restriction working life can not be brought into play the breeding advantage better, and outstanding individuality can not continue to serve breeding work owing to age limit.Clone technology has given to preserve fine germplasm resources, the new dawn of the continuity excellent individual length of service.But the nuclear transplantation efficient of pig is also very low, has not only hindered manufacturing speed and the scale of disease model, has also restricted the extensive clone of some good pig kinds.
Clone technology is the process of a more complicated, and body-cell neucleus transplanting is the basis of clone technology.Several factors affects success and the efficient of nuclear transplantation.Studies show that dissimilar donorcellses can provide different developmental potentialities, affect the growth efficient of clone embryos.For example, cumulus cell, embryonic stem cell-like, original kidney cell etc. can improve the efficient of pig nuclear transplantation.The cell of these types is positioned at growth status early, may provide apparent environment and the nucleus state of reprogrammed comparatively easily for nuclear transplantation.But the separation of these cells can damage health even the life of donor unavoidablely, can't promote aborning.
Mescenchymal stem cell (mesenchymal stem cells, MSC) also earlier on the growth status, have multipotency.The mescenchymal stem cell of pig can come out from marrow in separation and purification, and is little to the body damage of pig, and it is convenient to cultivate, and cost is less, therefore is the desirable donorcells source that adult pig carries out nuclear transplantation.There is research to report that the mescenchymal stem cell of pig can be finished the structure of clone embryos, still, also unsatisfactory to the raising degree of nuclear transplantation efficient.Therefore, also need this comparatively desirable nuclear transplantation donorcells is transformed and modified.
In addition, (induced pluripotent stem cell, IPSc) since the discovery of technology, the reprogrammed factor becomes " sharp weapon " that cell has versatility again to induce the reprogrammed stem cell since 2006.The reprogrammed factor is tended to change the apparent state of cell and the nuclear structure of cell, allows cell be in to be easier to the state of reprogrammed, promotes cell in the versatility differentiation potential of growing on the status.There is report to show that the donorcells of the single reprogrammed factor of overexpression Oct4 can improve the efficient of nuclear transplantation in cattle, improves the quality of ox clone embryos.Therefore, it is easier of the state of reprogrammed that the reprogrammed factor may help to allow mescenchymal stem cell be in, thereby improve the clone embryos reprogramming efficiency after the mescenchymal stem cell nuclear transplantation, promote clone pig embryo's growth efficient and quality, the convenient extensive clone who satisfies in the actual production the outstanding pig kind of growing up.
Summary of the invention
Technical problem to be solved by this invention is to overcome the inefficient defective of porcine clone embryos ectogenesis in the prior art, and a kind of method that improves porcine clone embryos ectogenesis efficient is provided.
To achieve these goals, the present invention is achieved by the following technical solutions:
A kind of method that improves porcine clone embryos growth efficient comprises the steps:
S1. separation and purification Medulla Sus domestica mescenchymal stem cell;
S2. Oct4, Klf4, c-Myc and 4 kinds of reprogrammed factors of Sox2 or Oct4, Klf4, c-Myc, Lin28, Nanog and 6 kinds of reprogrammed factors of Sox2 are changed over to and carry out overexpression in the Medulla Sus domestica mescenchymal stem cell; Utilize streaming fluorescent screening technical point to select the Medulla Sus domestica mescenchymal stem cell that contains 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 ~ 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
Stretching more (Shinya Yamanaka) professor in the mountain of Kyoto Univ Japan in 2006 utilizes retrovirus that four kinds of reprogrammed factors " Oct3/4; Sox2; c-Myc; Klf4 " are imported to have broken up completely in the mouse fibroblast first, with its again layout become totipotent class embryonic cell, proved that the reprogrammed factor has the reprogrammed effect, can change the destiny of cell.The reprogrammed factor just begins to be subjected to showing great attention to of academia.But former study finds that just the reprogrammed factor has the reprogrammed effect, but does not find that the reprogrammed factor can improve fetal development efficient.The invention provides a kind of novel donorcells, this cell is with specific reprogrammed factor modified; In addition, the present invention finds that first the Medulla Sus domestica mescenchymal stem cell that utilizes 4 kinds of reprogrammed factors (Oct4, Klf4, c-Myc and Sox2) or 6 kinds of reprogrammed factors (Oct4, Klf4, c-Myc, Lin28, Nanog and Sox2) to modify can improve nuclear transplantation efficient.At last, discovering that originally the reprogrammed factor is modified and prolongation is cultivated fate improving on the nuclear transplantation efficient, is discrepant.The Medulla Sus domestica mescenchymal stem cell that preferably, will contain 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 days or 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.The Medulla Sus domestica mescenchymal stem cell that more preferably, will contain 4 kinds of reprogrammed factors; Vitro culture 3 days or 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.The Medulla Sus domestica mescenchymal stem cell that most preferably, will contain 4 kinds of reprogrammed factors; Vitro culture 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
Preferably, behind separation and purification Medulla Sus domestica mescenchymal stem cell; Can pass through 6 passage streaming technology identification of cell purity, obtain negative rate and be lower than 2%, positive rate carries out genetic modification after being higher than 95% cell again.
Particularly, described raising porcine clone embryos is grown the method for efficient, comprises the steps:
S1. separation and purification Medulla Sus domestica mescenchymal stem cell; Utilize the expression of 6 kinds of antigens of CD14-APC-780, CD34-FITC, CD45-PE-Cy7, CD73-percp710, CD90-PE and 6 kinds of antibody test cell surfaces of CD105-APC, by 6 passage streaming technology identification of cell purity, obtain negative rate and be lower than 2%, positive rate is higher than 95% cell;
S2. the method that transforms by electricity changes Oct4, Klf4, c-Myc and 4 kinds of reprogrammed factors of Sox2 or Oct4, Klf4, c-Myc, Lin28, Nanog and 6 kinds of reprogrammed factors of Sox2 over to and carries out overexpression in the Medulla Sus domestica mescenchymal stem cell; Utilize streaming fluorescent screening technical point to select the Medulla Sus domestica mescenchymal stem cell that contains 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 ~ 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
Compared with prior art, the present invention has following beneficial effect:
1, (porcine embryonic fibroblast PEF) compares, and pMSC can improve the developmental rate 5.79%(74.23% of clone embryos as cell donor with conventional cell donor pig embryo fibroblast
Vs. 80.02%,
P=0.14), improves the blastaea rate 18.43%(20.15% of clone embryos
Vs. 38.58%,
P＜0.05), increases the cell count 15.8(41.60 of clone embryos blastula stage
Vs. 54.7,
P=0.225).
2, do not compare with containing the pMSC cell donor that RFs modifies, 4RFs-7days-pMSC and 6RFs-7days-pMSC cell can significantly improve the division efficient of clone embryos as donor, have improved 9.60%(respectively
P＜0.05), 11.57%(
P＜0.05).
3, compare with the pMSC cell donor that does not contain the RFs modification, the 4RFs-3days-pMSC cell can significantly improve the blastaea rate 7.31%(of clone embryos as donor
P＜0.05).
4, compare with the pMSC cell donor that does not contain the RFs modification, 4RFs-3days-pMSC, 4RFs-7days-pMSC, 6RFs-3days-pMSC, 6RFs-7days-pMSC cell can increase cell count blastula stage of clone embryos as donor, have increased 4.5(respectively
P=0.414), 10.25(
P=0.083), 2.25(
P=0.742), 9(
P=0.265).
5, more above-mentioned 5 class cell donors to the raising degree of nuclear transplantation efficient as can be known, 4RFs-7days-pMSC not only can help the pMSC of unmodified improve division rate and blastula stage cell count, can also significantly help the pMSC of unmodified to realize higher blastaea rate.
Figure of description
Fig. 1. experiment flow synoptic diagram of the present invention.
Fig. 2. the Medulla Sus domestica mescenchymal stem cell photo that separation and purification is come out and growth curve measurement result; A. cultivate 3 days state; B. cultivate 5 days state; C. cultivate 10 days state; D. the state that goes down to posterity and cultivate; E. the growth curve in 7 of cell days.
Fig. 3. the groupization method detects the Medulla Sus domestica mescenchymal stem cell and becomes fat, skeletonization, one-tenth cartilage differentiation capability; A. inducing cell becomes the control group oil red coloration result of fat; B. the control group sodium alizarinsulfonate coloration result of inducing cell skeletonization; C. the control group alcian blue coloration result of inducing cell skeletonization; D. inducing cell becomes the experimental group oil red coloration result of fat; E. the control group sodium alizarinsulfonate coloration result of inducing cell skeletonization; F. inducing cell becomes the experimental group alcian blue coloration result of fat.
Fig. 4. the expression of the 6 kinds of antigens in the disposable evaluation Medulla Sus domestica of cell streaming method mescenchymal stem cell surface; A. the cell of Fen Xiing is established door; B is jack to jack adapter 2 passage detected results; C is the negative 2 passage detected results of Dan Yangdan; The two positive 2 passage detected results of D; EFG is single negative single passage detected result; HIJ is single positive single passage detected result.
Fig. 5. the Medulla Sus domestica mescenchymal stem cell is carried out the reprogrammed factor modify and results of screening; A. the cell state behind the electrotransfection reprogrammed factor OSKM under the light field; B. the cell state behind the electrotransfection reprogrammed factor OSKM of fluorescence-activation; C. the cell state behind the electrotransfection reprogrammed factor OSKMLN under the light field; D. the cell state behind the electrotransfection reprogrammed factor OSKMLN of fluorescence-activation; E. the thin method of streaming is assessed the result of electrotransfection reprogrammed factor OSKM efficient; F. the thin method of streaming is assessed the result of electrotransfection reprogrammed factor OSKMLN efficient; G. the fluorescence fluidic cell screens the cell colony door of setting before the cell; H. the fluorescence fluidic cell screens the cell door of the separation 4RFs-pMSC that sets before the cell; I. the 4RFs-pMSC result that is sorted into of fluorescence fluidic cell sieve method; J. cell door and the separation results of the sorting 6RFs-pMSC that sets of fluorescence fluidic cell sieve method; K. the 4RFs-pMSC cultivation results that is sorted into; L. the 6RFs-pMSC cultivation results that is sorted into.
Fig. 6. the Medulla Sus domestica mescenchymal stem cell is to the impact evaluation result of body outer clone fetal development efficient; A. the clone embryos produced as donorcells of control group pEF; B. the clone embryos produced as donorcells of experimental group pMSC; C. the nuclear staining result in control group pEF clone embryos stage blastula stage of producing as donorcells; D. the nuclear staining result in experimental group pMSC clone embryos stage blastula stage of producing as donorcells; E. pEF and pMSC are as the comparative result of donorcells to clone embryos division efficient; F. pEF and pMSC form the comparative result of efficient to the clone embryos blastaea as donorcells; G. pEF and pMSC are as the comparative result of donorcells to clone embryos total cellular score blastula stage.
Fig. 7. the Medulla Sus domestica mescenchymal stem cell that the reprogrammed factor is modified is to the impact evaluation result of body outer clone fetal development efficient; A. pMSC, 4RFs-3days pMSC, 6RFs-3days pMSC divide the comparative result of efficient, blastaea formation efficient respectively to clone embryos as donorcells; B. pMSC, 4RFs-3days pMSC, 6RFs-3days pMSC are respectively as the comparative result of donorcells to clone embryos total cellular score blastula stage; C. pMSC, 4RFs-7days pMSC, 6RFs-7days pMSC divide the comparative result of efficient, blastaea formation efficient respectively to clone embryos as donorcells; D. pMSC, 4RFs-7days pMSC, 6RFs-7days pMSC are respectively as the comparative result of donorcells to clone embryos total cellular score blastula stage; E. pMSC, 4RFs-3days pMSC, 6RFs-3days pMSC, 4RFs-7days pMSC, 6RFs-7days pMSCF promote the comparative result of effect respectively to the clone embryos division rate as donorcells; F. pMSC, 4RFs-3days pMSC, 6RFs-3days pMSC, 4RFs-7days pMSC, 6RFs-7days pMSCF promote the comparative result of effect respectively to clone embryos blastaea rate of formation as donorcells.
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.Employed test method is ordinary method if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are reagent and the material that can obtain from commercial channels.
Embodiment 1
One, material
α MEM nutrient solution is available from Life Technologies company; Reagent is available from sigma company; Antibody is available from eBioscience company; Plasmid pLv-CMV-ZsGreen-hOct4(O, carry people Oct4 gene order, Genbank:NM_002701.4), pLv-CMV-ZsGreen-hKlf4(K, carry people Klf4 gene order, Genbank:NM_004235.4), pLv-CMV-ZsGreen-hc-Myc(M, carry people c-Myc gene order, Genbank:NM_002467.4), pLv-CMV-ZsGreen-hLin28(L, carry people Lin28 gene order, Genbank:NM_024674.4), pLv-CMV-ZsGreen-hNanog(N, carry people Nanog gene order, Genbank:NM_024865.2), pLv-CMV-ZsGreen-hSox2(S, carrying people Sox2 gene order, Genbank:NM_003106.3) is the animal genetic engineering laboratory preservation of life science institute of Zhongshan University.
Two, testing sequence: whole schematic flow sheet of the present invention is seen as shown in Figure 1.
S1. separation and purification obtains mesenchymal stem cells MSCs
S11. separate marrow:
Extract shin of pork marrow 5 ~ 10mL, after heparin sodium and the physiological saline dilution, (the erythrocyte lysate is formed: 10 * storage liquid, 1.5M NH to add the erythrocyte lysate
4Cl, 100mM KHCO
3, 1mM EDTA, after 0.22 μ m filters ,-20 ℃ of preservations) room temperature placement 5min, the centrifugal 10min of 1600rpm, collecting cell precipitation; After cell precipitation α MEM nutrient solution (α MEM nutrient solution is formed: 20% FBS, 2mM L-glutamax, 1% NAA, 50Unit/mL penicillin, 50 μ/mL streptomycin) washing, recentrifuge.
S12. separation and purification cell:
After the cell of step S11 acquisition is resuspended with α MEM nutrient solution, according to 1 * 10
6/ cm
2Ratio be inoculated in the plastic culture dish, place 5%CO
2, cultivate under 37 ℃ of conditions; After cultivating 12h, rock nutrient solution gently, remove nutrient solution then fully, add fresh medium and continue to cultivate; After cultivating 24h, rock nutrient solution gently, half amount removes nutrient solution then, adds fresh medium and continues to cultivate; Continue culturing cell 5 ~ 10days, the one-tenth fibrous cell of appearance is the mesenchymal stem cells MSCs of pig; Shown in the A among Fig. 2, B, C, D.
S13. mesenchymal stem cells MSCs is identified:
(1) growth curve is measured: the mesenchymal stem cells MSCs of step S12 purifying is inoculated on 96 orifice plates (2000/ hole) cultivates, after adherent, PBS washes 2 times, add and contain in the nutrient solution of Alamar blue, continue to cultivate 8 days, set time every day is detected 570nm, 600nm light absorption value, calculates then, analyzes, draw growth curve, the result is shown in the E among Fig. 2.
(2) cell cultures to 100% is converged after, add into fat respectively, osteogenic induction liquid carries out 21 days cultivation.Become fat induced liquid prescription: MEM alpha (Gibico), FBS 20% (PAA), 2mM L-Glumax (Gbico), 1% Non Essential Amino Acid, 3 – isobutyl-1-methylxanthine (IBMX) (0.5mM), Dexamethasone (0.25 μ M), Insulin (50 μ g/mL); Osteogenic induction liquid formula: MEM alpha (Gibico), FBS 20% (PAA), Glumax (Gbico), Non Essential Amino Acid, β-Glycerophosphate disodium salt hydrate (10 mM), Dexamethasone (100nM), Acorbic acid (50 μ g/mL), be detected as fat, skeletonization effect with oil red O, sodium alizarinsulfonate dyeing respectively then, the results are shown in Figure 3, isolated cells can form red fat granule and contain calcareous bone tubercle.Cell harvesting after will converging becomes chondrocyte induction to cultivate in centrifuge tube.Chondrocyte induction liquid formula: MEM alpha (Gibico), FBS 20% (PAA), Glumax (Gbico), Non Essential Amino Acid, TGF-β (10ng/mL) (R﹠amp; D), Dexamethasone (100nM), Acorbic acid (50 μ g/mL), ITS+1 (Gibico) detected with the alcian blue dyeing process after 21 days, the F among result such as Fig. 3, isolated cells can form the cartilaginous tissue that contains the cartilage mucopolysaccharide.Show that separating the pMSC that obtains maintains developmental potentiality preferably.
(3) utilize different fluorescently-labeled antibody (CD14-APC-780, CD34-FITC, CD45-PE-Cy7, CD73-percp710, CD90-PE, CD105-APC), hatch the mesenchymal stem cells MSCs of pig, utilize 6 passage flow cytometers to identify the mesenchymal stem cells MSCs of the pig that separation and purification is come out then; Hyperchannel streaming authentication method:
1) after collecting cell: PBS washes 2 times, after 0.25% trysinization, 1600rpm * 5min collecting cell;
2) antibody incubation: cell is blown and beaten into 1 * 10 gently with PBS
6/ mL single cell suspension is distributed into the 1mL/ pipe; Add 5 ~ 10 μ L CD105, CD34, CD45, CD90, CD73, CD14 primary antibodie respectively, negative control, blank are set simultaneously, fully piping and druming is even, and lucifuge is hatched 30 min on ice;
3) cell fixation: 2000 r/min, centrifugal 10min, washing lotion (form: 2%BSA, PBS, 0.1% NaN by washing lotion
3) wash 2 times, add the Paraformaldehyde 96 stationary liquid 300 μ L of 4%, pH 7.4;
4) streaming detects: behind the 300 order cells sieve filtration cell, the upflowing cell instrument is analyzed; Earlier with blank voltage is set, the cell colony door draws; Re-use the standard substance that have different fluorescence and carry out parameter testing and fluorescence compensation; Test experience group sample utilizes and with the FlowJ analytical system detected result is analyzed, drawn then.The results are shown in Figure 4.Isolated cells shows CD14, CD34, CD45 feminine gender, CD73, CD90, the CD105 positive, and negative rate is lower than 2% of international artery cell association regulation simultaneously, and positive rate is higher than 95% of standard code.Show that separating the pMSC that obtains is the cell colony of comparison homogeneous.
S2. mesenchymal stem cells MSCs is carried out genetic modification
S21. electricity changes cell:
The method of utilizing electricity to change, with 4 kinds of reprogrammed factors (Oct4, Klf4, c-Myc, Sox2) or 6 kinds of the reprogrammed factors (Oct4, Klf4, c-Myc, Lin28, Nanog, Sox2) change the mesenchymal stem cells MSCs of pig respectively over to, the Medulla Sus domestica mescenchymal stem cell that electricity changes into merit will carry green fluorescence labelled protein (ZsGreen); Concrete electricity changes step:
(1) prepare cell: when mesenchymal stem cells MSCs is cultured to 80 ~ 90% degree of converging, after the trysinization, centrifugal collecting cell; It is resuspended to change liquid with 100 μ L electricity.
(2) prepare electricity and change reagent: respectively 4 kinds of plasmids of pLv-CMV-ZsGreen-hOct4, pLv-CMV-ZsGreen-hKlf4, pLv-CMV-ZsGreen-hc-Myc, pLv-CMV-ZsGreen-hSox2 of 1 μ g/ μ L and 6 kinds of plasmids of pLv-CMV-ZsGreen-hOct4, pLv-CMV-ZsGreen-hKlf4, pLv-CMV-ZsGreen-hc-Myc, pLv-CMV-ZsGreen-hLin28, pLv-CMV-ZsGreen-hNanog, pLv-CMV-ZsGreen-hSox2 of 1 μ g/ μ L are added in the enchylema, mixing, room temperature is placed 3min;
(3) electricity changes: the electricity that Nero nucleus electroporation is set changes parameter, 1700V, and 20ms, 1plus, the tips that makes electricity consumption change what 100 μ L of rifle carries out electricity to be changeed.
(4) cultivate: the cell after electricity changes continues to cultivate in fresh nutrient solution, changes liquid behind the 12h.
(5) identify: utilize cell streaming technology for detection cell fluorescence intensity, carry out cell with reference to blank cell and establish door; Change fluorescence intensity according to the single plasmid of cell electricity then, the cell that the screening high fluorescent is set is positive cell, as Fig. 5 BD, utilizes the airflow classification technology screening to go out to have cell than hyperfluorescence intensity.
S22. screen cell:
Utilize the streaming fluorescent screening process, isolate the Medulla Sus domestica mescenchymal stem cell that carries 4 kinds of reprogrammed factors (Oct4, Klf4, c-Myc, Sox2), 6 kinds of reprogrammed factors (Oct4, Klf4, c-Myc, Lin28, Nanog, Sox2) respectively; Continuation is at 5%CO
2, cultivated 2 ~ 6 days under 37 ℃ of conditions; Streaming fluorescent screening cell step:
(1) after collecting cell: PBS washes 2 times, after 0.25% trysinization, 1600rpm * 5min collecting cell;
(2) antibody incubation: cell is blown and beaten into 1 * 10 gently with PBS
6/ mL single cell suspension; Behind the 300 order cells sieve filtration cell, the upflowing cell instrument is analyzed;
(4) streaming detects: with blank voltage is set earlier, the cell colony door draws; The reference substance that re-uses the fluorescence that has single plasmid carries out parameter testing; Test experience group sample is adjusted purpose cell door (as Fig. 5 GHIJ) then, filters out the positive cell of high fluorescent, proceeds to cultivate, as Fig. 5 KL.The cell that screening obtains is that 4RFs-3days(namely cultivates the Medulla Sus domestica mescenchymal stem cell that contained 4 kinds of reprogrammed factors in 3 days), 6RFs-3days(namely cultivates the Medulla Sus domestica mescenchymal stem cell that contained 6 kinds of reprogrammed factors in 3 days), 4RFs-7days(namely cultivates the Medulla Sus domestica mescenchymal stem cell that contained 4 kinds of reprogrammed factors in 7 days), 6RFs-7days(namely cultivates the Medulla Sus domestica mescenchymal stem cell that contained 6 kinds of reprogrammed factors in 7 days).
S3. make clone embryos
Step S2 is cultivated the 4RFs-3days that obtains, 6RFs-3days, 4RFs-7days, 6RFs-7days Medulla Sus domestica mescenchymal stem cell carries out the making of porcine clone embryos as the nuclear donor cell, also makes clone embryos with the pEF cell with without the pMSC cell of genetic modification as donorcells simultaneously.
S31. recipient cell is prepared: after separating ovary, collect ovocyte, put into 0.5 μ g/mL follicular stimulating hormone (follicle-stimulating hormone, FSH), 0.5 μ g/mL lutropin (Luteinizing hormone, LH), 10ng/ml Urogastron (epidermal growth factor, EGF), 10% pig follicle liquid (porcine follicular fluid, PFF) and nutrient solution (TCM-199, the 26.19mM NaHCO of 0.57mM halfcystine
3, 3.05mM D-glucose, 0.91mM Sodium.alpha.-ketopropionate, 75 μ g/mL penicillin sodium salts, 50 μ g/mL streptomycin sulfates, 0.1% PVA) and the middle 22h that cultivates; In not containing the above-mentioned substratum of hormone and halfcystine, cultivate 22h again; Handle with 0.1% Unidasa again and remove cumulus cell, put into embryo operation liquid and be made into drop, be placed under the mineral oil.
S32. ovocyte stoning: selection has polar body, the uniform mature oocyte of tenuigenin is placed on the microinjection operator's console, uses blind suction method to remove polar body.(locking pin mouth external diameter is 150 ~ 200 μ m, and internal diameter is 30 ~ 50 μ m; Kernel removing needle mouth external diameter is 20 ~ 30 μ m, and internal diameter is 6 ~ 10 μ m, 45 ° of angles)
S33. injection nuclear transplantation in the tenuigenin: will digest the notes nuclear operation liquid (TCM-199 that the donorcells of collecting moves into the micrurgy dish, HEPES) drip, draw donorcells about 15 μ m with kernel removing needle, at the otch that zona pellucida stays donorcells is directly injected in the tenuigenin of ovocyte during then along stoning, finish the structure of clone embryos.Use ECM electricity fusion instrument, the electric pulse of the 1.2kV/cm 30 μ sec that shock by electricity.
S34. embryo culture: clone embryos is transferred to PZM-5(Porcine zygote medium 5) in cultivate, during 36 ~ 48h and 6 days embryo number and the blastaea numbers of counting division under Stereo microscope respectively, and calculate division rate and blastaea rate of formation; Utilize the cell count of DAPI dyeing counting blastula embryo.
Interpretation of result:
Division rate: the 4RFs-3days of cultivation, 6RFs-3days pMSC all can improve division rate as the nuclear donor cell, but not statistically significant; The 4RFs-7days that cultivates, 6RFs-7days pMSC all can improve division rate as the nuclear donor cell, have statistical significance (
P＜0.05).As Fig. 7 A.
The blastaea rate: the 6RFs-3days pMSC of cultivation all can improve division rate as the nuclear donor cell, but not statistically significant; The 4RFs-7days that cultivates, 6RFs-7days pMSC all can improve division rate as the nuclear donor cell, have statistical significance (
P＜0.05).As Fig. 7 C.
Cell count: though the pMSC of 6RFs-7days can increase the cell count of blastula embryo as the nuclear donor cell, make clone embryos produce maximum cell numbers, but, the cell count SD(Std. Deviation of the pMSC of 4RFs-7days) value is minimum, show that clone embryos is in the homogeneity state than the homogeneous cell quantity, as Fig. 7 D.
S4. judge and improve fetal development efficient
S41 pMSC can improve clone embryos and grow efficient:
When the pMSC that uses not gene mentation modification made clone embryos as donorcells, the result showed that with respect to traditional cell donor pEF cell, the division rate of pMSC clone embryos, blastaea rate and cell count have raising.As Fig. 6.Separate the mescenchymal stem cell that obtains and show good developmental potentiality, can significantly improve the growth efficient of clone embryos, improve embryo's division rate and blastula embryo cell count.This helps to realize:
(1) obtains outstanding donorcells from the pig body to minimum infringement.Because PEF derives from brephic body, greatly damaged pregnant pig health, damaged fetus; And the pMSC of derived from bone marrow can reduce the damage to body, and stress reaction is little, and is easy and simple to handle.
(2) be more suitable in the clone of the pig kind of growing up.Because pMSC can obtain from marrow blood by the simple operation step; and the cell that once can obtain sufficient amount is used for purifying, cultivation and evaluation; training method is simple, cultivate require simple and easy, the clone operations of the mass-producing of very convenient adult individuality.
The pMSC that the S42 reprogrammed factor is modified can improve clone embryos better and grow efficient:
Because the stem cell clone of pig is not successfully set up yet, the performance that the IPS cell shows aspect nuclear transplantation is barely satisfactory.Use the reprogrammed factor further to improve the clone embryos structuring capacity of pMSC, not only can help to realize higher division rate, blastaea rate, cell count, further improve clone embryos and grow quality, can also establish good basis for embryo transfer.And as cell donor, the pMSC that contains the reprogrammed factor more can further improve the division rate of clone embryos with respect to pMSC.
(1) along with the increase of the reprogrammed factor, the division rate of clone embryos has obtained further raising, and as Fig. 7 E, 6RFs more can improve the division rate of clone embryos than 4RFs pMSC.
(2) along with the increase of cultivating fate, the division rate of clone embryos has obtained significant raising, as Fig. 7 E, 4RFs-7days than 4RFs-3days pMSC more can significantly improve clone embryos division rate (
P＜0.05).
(3) still, along with the increase of the reprogrammed factor and the increase of cultivating fate, the blastaea rate of clone embryos but is not significantly improved; As Fig. 7 F.
(4) yet, along with the increase of the reprogrammed factor, the cell count of blastula embryo is improved; Though there is not the meaning on the statistics, 4RFs-7days pMSC can obtain cell count comparatively uniformly.As Fig. 7 D.
(5) based on the above results, 4RFs-7days pMSC is more suitable for the donorcells as nuclear transplantation, can obtain higher growth quality and homogeneous clone embryos.
Claims (6)
1. one kind is improved the method that porcine clone embryos is grown efficient, it is characterized in that, comprises the steps:
S1. separation and purification Medulla Sus domestica mescenchymal stem cell;
S2. Oct4, Klf4, c-Myc and 4 kinds of reprogrammed factors of Sox2 or Oct4, Klf4, c-Myc, Lin28, Nanog and 6 kinds of reprogrammed factors of Sox2 are changed over to and carry out overexpression in the Medulla Sus domestica mescenchymal stem cell; Utilize streaming fluorescent screening technical point to select the Medulla Sus domestica mescenchymal stem cell that contains 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 ~ 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
2. grow the method for efficient according to the described raising porcine clone embryos of claim 1, it is characterized in that, contain the Medulla Sus domestica mescenchymal stem cell of 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 days or 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
3. grow the method for efficient according to the described raising porcine clone embryos of claim 2, it is characterized in that, contain the Medulla Sus domestica mescenchymal stem cell of 4 kinds of reprogrammed factors; Vitro culture 3 days or 7 days; Namely can be used as the nuclear donor cell and carry out nuclear transplantation.
4. grow the method for efficient according to the described raising porcine clone embryos of claim 3, it is characterized in that, contain the Medulla Sus domestica mescenchymal stem cell of 4 kinds of reprogrammed factors; Vitro culture 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
5. grow the method for efficient according to the described raising porcine clone embryos of claim 1, it is characterized in that, behind the described separation and purification Medulla Sus domestica of the S1 mescenchymal stem cell; By 6 passage streaming technology identification of cell purity, obtain negative rate and be lower than 2%, positive rate carries out genetic modification after being higher than 95% cell again.
6. grow the method for efficient according to the described raising porcine clone embryos of claim 1, it is characterized in that, comprise the steps:
S1. separation and purification Medulla Sus domestica mescenchymal stem cell; Utilize the expression of 6 kinds of antigens of CD14-APC-780, CD34-FITC, CD45-PE-Cy7, CD73-percp710, CD90-PE and 6 kinds of antibody test cell surfaces of CD105-APC, by 6 passage streaming technology identification of cell purity, obtain negative rate and be lower than 2%, positive rate is higher than 95% cell;
S2. the method that transforms by electricity changes Oct4, Klf4, c-Myc and 4 kinds of reprogrammed factors of Sox2 or Oct4, Klf4, c-Myc, Lin28, Nanog and 6 kinds of reprogrammed factors of Sox2 over to and carries out overexpression in the Medulla Sus domestica mescenchymal stem cell; Utilize streaming fluorescent screening technical point to select the Medulla Sus domestica mescenchymal stem cell that contains 4 kinds of reprogrammed factors or 6 kinds of reprogrammed factors; Vitro culture 3 ~ 7 days, namely can be used as the nuclear donor cell and carry out nuclear transplantation.
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