CN108588008A - A kind of black pig handmade cloning in Debao reconstructs drug and the application of vitro Development of Embryos - Google Patents
A kind of black pig handmade cloning in Debao reconstructs drug and the application of vitro Development of Embryos Download PDFInfo
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- CN108588008A CN108588008A CN201810349338.6A CN201810349338A CN108588008A CN 108588008 A CN108588008 A CN 108588008A CN 201810349338 A CN201810349338 A CN 201810349338A CN 108588008 A CN108588008 A CN 108588008A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
Abstract
The invention belongs to gene engineering technology fields, disclose drug and the application of a kind of black pig handmade cloning reconstruct vitro Development of Embryos in Debao, it is found after carrying out 5 Aza CdR processing 72h to donor fibroblast, 5 Aza CdR of suitable concentration (0.25 μm of ol/L~0.5 μm ol/L) can reduce the fibroblastic methylation level of donor nuclei, and improve blastocyst rate and blastaea total cell number, various concentration and 5 Aza CdR of different time is used to handle reconstructed embryo respectively, count its cleavage rates, blastocyst rate and blastomere number, as a result, it has been found that 20nmol/L, 72h is optimal treatment condition.Due to after handling in succession donor and reconstructed embryo, the medicament residue and its cytotoxicity of 5 Aza CdR itself can show the effect of superposition on reconstructed embryo for analysis, it is determined that analysis method is to reaching optimum efficiency.
Description
Technical field
The invention belongs to the black pig handmade clonings of gene engineering technology field more particularly to a kind of Debao to reconstruct embryoid body outgoing
The drug educated and application.
Background technology
Currently, the combination of body-cell neucleus transplanting (SCNT) technology and genomics modification technique, is transgenic animals
Production, human gene disease the research of the fundamental biological knowledges such as development, the xenotransplant of animal model provide and have by force
The technical support of power.Since pig is closely similar in dissection, tissue, physiology, Nutrition and Metabolism etc. and the mankind, so being considered as
The optimal donor of mankind's Xenogeneic organ's nuclear transfer, then scientist both domestic and external eye is focused on into SCNT technologies and base one after another
Because of the combination of group modification technique.Due to big domestic animal especially pig cloning efficiency still very low (1%~2%), greatly restrict
The development of somatic cell clone pig and transgene clone pig correlative study work.Influence somatic cell clone reconstructed embryo early development effect
Fruit it is many because being known as, such as donorcells and recipient cell growth cycle coordination, the process of caryoplasm fusion, and develop and arrive
Can the DNA methylation of nucleocytoplasmic exchange and reconstructed embryo and demethylation level etc. are smoothly completed after the 8-cell stages influences
To the normal development of body-cell neucleus transplanting reconstructed embryo.Carrying out epigenetic modification processing to donorcells and reconstructed embryo can improve
The potentiality of development of nuclear transfer embryo.Dnmt rna inhibitor be the epigenetic modification that is widely used in body cell because
Son, it methylates with demethylation in SCNT, has decisive role to donor and the reprogramming of acceptor gene group.5- nitrogen
Miscellaneous -2 '-deoxycytidine (5-Aza-2 '-deoxycytidine, 5-Aza-CdR) is that irreversible dnmt rna inhibits
Agent, with the cytotoxicity caused double effect of low dosage activation methylation silenced genes, high dose.It is recognized by U.S. FDA
Card is clinically one of common anticancer drug, while it is used as DNA methylation enzyme inhibitor by people.Show to use
5-Aza-CdR processing donorcells can improve the potential that nuclear transplantation in cattle embryo develops in vitro.But the 5-Aza-CdR of high concentration
Also it is verified that having cytotoxicity to nuclear transplantation in cattle embryo, and the 5-Aza-CdR of low concentration processing donorcells are beneficial to
The development of porcine clone embryos.It is found after handling fibroblast donor first with 5-Aza-CdR by research, high dose 5-
Aza-CdR can reduce donorcells and the DNA methylation of reconstructed embryo is horizontal, but have inhibition to the development of embryo, after
To find that low dosage can achieve the purpose that reduce methylation level while improving blastaea to develop.It is black that 5-Aza-CdR handles Debao
Pig HMC reconstructed embryos, traditional analysis method can not determine the matched proportion density of 5-Aza-CdR, can not understand it to reconstructing outside idiosome
The influence of potentiality of development.
In conclusion problem of the existing technology is：5-Aza-CdR handles the black pig HMC reconstructed embryos in Debao, traditional point
Analysis method can not determine the matched proportion density of 5-Aza-CdR；If 5-Aza-CdR dosage is excessively high can to reduce donorcells and reconstructed embryo
DNA methylation it is horizontal, but have inhibition, analysis method that can not understand it to being developed outside reconstruct idiosome the development of embryo
The influence of potential.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of black pig handmade clonings in Debao to reconstruct embryoid body outgoing
The drug educated and application,
The invention is realized in this way a kind of drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao, the moral
The drug for protecting black pig handmade cloning reconstruct vitro Development of Embryos is 5-aza-2'-deoxycytidine；
0.25 μm of ol/L~0.5 μm ol/L of the 5-aza-2'-deoxycytidine concentration；Handle 72h.
Further, a concentration of 20nmol/L of the 5-aza-2'-deoxycytidine handles 72h.
Another object of the present invention is to provide the medicines that a kind of black pig handmade cloning in Debao reconstructs vitro Development of Embryos
Application of the object in the black pig cultivation in Debao.
The present invention after donor fibroblast progress 5-Aza-CdR processing 72h to having found, suitable concentration (0.25 μm of ol/L
~0.5 μm of ol/L) 5-Aza-CdR can reduce the fibroblastic methylation level of donor nuclei, and improve blastaea hair
Educate rate and blastaea total cell number, various concentration and different time 5-Aza-CdR used to handle reconstructed embryo respectively, count its cleavage rates,
Blastocyst rate and blastomere number, as a result, it has been found that 20nmol/L, 72h are optimal treatment condition.Analysis is due in succession to donor
After being handled with reconstructed embryo, the medicament residue and its cytotoxicity of 5-Aza-CdR itself can show to be superimposed on reconstructed embryo
Effect, it is determined that analysis method is to reaching optimum efficiency.
Description of the drawings
Fig. 1 is 5-Aza-CdR provided in an embodiment of the present invention on reconstructing in the analysis method that vitro Development of Embryos influences not
With the display figure of HMC blastaeas after concentration 5-Aza-CdR processing reconstructed embryos.
Fig. 2 is the analysis method difference that 5-Aza-CdR provided in an embodiment of the present invention influences reconstruct vitro Development of Embryos
The display figure of HMC blastaeas Hoechst33342 dyeing after concentration 5-Aza-CdR processing reconstructed embryos.
Fig. 3 is the analysis method 5- that 5-Aza-CdR provided in an embodiment of the present invention influences reconstruct vitro Development of Embryos
Aza-CdR handles the display figure of HMC blastaeas Hoechst33342 dyeing after reconstructed embryo different time.
Fig. 4 is the analysis method difference that 5-Aza-CdR provided in an embodiment of the present invention influences reconstruct vitro Development of Embryos
Concentration 5-Aza-CdR handles the display figure of HMC blastaeas Hoechst33342 dyeing after donor and reconstructed embryo simultaneously.
Specific implementation mode
In order to further understand the content, features and effects of the present invention, the following examples are hereby given, and coordinate attached drawing
Detailed description are as follows.
The application principle of the present invention is further described with reference to experiment.
1 materials and methods
1.1 main agents consumptive materials
Fetal calf serum (FBS) is purchased in Gbico companies, and TCM-199 pulvis, DMEM pulvis are purchased in Gbico companies of the U.S.,
Hormone is eCG, and hCG is purchased in Sigma Co., USA, remaining chemical reagent is purchased unless otherwise specified in U.S. Sigma
Aldrich.Cell fusion apparatus model：ECM2001, integration slot model：BTX 450, embryo gather needle-like number：DN-10 formulas,
Oocyte maturation culture dish：35mm and 60mm plastics plates, stoning cutter self-control, Embryo Culture ware：Four orifice plates.
The maturation culture of 1.2 egg mother cells
Ovary is obtained from the slaughterhouse of Nanning City, is fitted into containing in dual anti-physiological saline, is sent within 4h with thermo jug
Go back to laboratory.First ovary is rinsed well with Normal Saline, then be added preheating containing dual anti-physiological saline, send embryo back to
Proloculum extracts egg mother cell.The ovarian follicle of 2~6mm of Ovarian surface is extracted with the syringe of 10mL, liquor folliculi is put into test tube and stands,
It discards supernatant, sediment fraction is stayed to dispense.Select has particle/cumulus cell coating, the good ovarian cumulus of refractivity containing having three layers or more
Oocyte complex (COCs) is put into ripe liquid (eCG of hCG+10IU/mL containing 10IU/mL) culture, is changed after 20~22h
At the culture solution without hormone, continue 18~20h of culture to cell maturation.
The culture (tissue mass cell culture) of 1.3 fetal fibroblasts
Tire pig obtains from Nanning City slaughterhouse, is placed in dual anti-physiological saline, sends laboratory in 4h back to.First use generic physiological
Brine cleans three times, is transferred to sterile super-clean bench, isolates part thigh tissue, is cleaned 2 times, put with containing dual anti-PBS later
The plate for entering 60mm is shredded to the tissue block of 2~3mm, and the DMEM cultures containing 10%FBS on a small quantity are added into culture dish
It is uniformly spread out with tweezers, performs label in ware lid, be buckled in incubator, a small amount of culture solution is added after 4h by liquid, is prevented
Only tissue block floats.Culture solution is filled it up in plate bottom after for 24 hours, continues to observe fibroblastic upgrowth situation after 48h, wait for into
It when fibroblast growth to 90% or so degree of converging, is passed on, is used as pig handmade cloning donorcells.
The pitting method of 1.4 egg mother cells
It selects good egg mother cell to be placed in egg-cleaning liquid, blow and beat for several times to remove granular cell.Then chain is moved it into
In fungi protease solution, when oolemma starts deformation, is moved into immediately containing the middle washing of washing lotion (T20) for 20%FBS, be then placed in
Medium its of T20 reverts to circle, and egg mother cell is transferred in cutting liquid (containing 2.5 μ g/mL CB) with glass pipette, is selected extensive
Multiple complete egg mother cell is for being enucleated.
Polar body positioning mode：Polar body, is placed in the position of or at 6 points at 12 points, uses cutter by the complete cell of picking first polar body
The ooecium matter in polar body direction about 1/3 is cut, stoning cytoplasm, which stays, does follow-up test.
Fusion/activation of 1.5 confessions/recipient cell
Using a step fusion method.Fusion/activation is divided into three steps, is bonded first, then carries out merging/electrically activating again,
Finally carry out chemokinesis.
Bonding：It first uses phytolectin (PHA) that seedless half ovum and donorcells are bonded, forms half ovum-donorcells and match
To object；Then again that another half ovum of stoning and counter pair is bonding, the adherend of ovum-donor-ovum formula is formed, electro' asion is moved into
It is to be fused in liquid.
Fusion/electrical activation：Adherend is uniformly put into the integration slot of electro' asion liquid, electrofusion parameter is：AC：6V/
cm；DC：1.2kV/cm, 30 μ s, 2DC.It is put into incubator after the completion of after electrical activation and restores 30min, then check that it merges feelings
Condition.
Chemokinesis：Reconstructed embryo after fusion is put into and is activated in liquid containing the PZM-3 of 5 μ g/mL CB and 10 μ g/mL CHX
Activation, often drop, which activates, is put into 1 piece of reconstructed embryo in liquid, activate 4h.
The micro-cavity culture of 1.6 reconstructed embryos
To ensure that the HMC reconstructed embryos of lensless Fourier transform holography can be grown under relatively independent environment, this research is trained using micro-cavity
The method of supporting.By the embryo transfer after activation in advance in CO2At least 4h is balanced in incubator and is had in the PZM-3 that paraffin oil covers
Micro-cavity in.The preparation of micro-cavity：Micro-cavity is pricked in four orifice plate bottom Rotating with Uniform with embryo aggregation needle, micro-cavity size is according to experiment
It is required that being arranged, 1 piece of embryo is put into each micro-cavity.48h observes cleavage rates, and 160~168h observes blastocyst rate.
1.7 blastomeres count
The blastaea for collecting the 6th~7 day, after being cleaned 2~3 times in CCM, 10 μm of ol/mL Hoechst33342 being placed in contaminate
Liquid is protected from light dyeing 15min in room temperature, is cleaned 2~3 times with CCM after taking-up, paraffin-vaseline mounting, seen under fluorescence microscope again
Examine record blastomere total number.
1.8 experimental design
Experiment one：Compare 40nmol/L, 20nmol/L, 10nmol/L, tetra- kinds of concentration 5-Aza- of 5nmol/L, 0nmol/L
CdR handles HMC and reconstructs embryo 72h, the influence to its developmental capacity；
Experiment two：Compare 20nmol/L 5-Aza-CdR processing HMC reconstruct embryo 96h, 72h, 48h, for 24 hours, 0h sends out it
Educate the influence of effect；
Experiment three：Compare 1 μm of ol/L, 0.5 μm of ol/L, 0.25 μm of ol/L, 0 μm of ol/L, tetra- processing groups and reconstructed embryo are most
Good concentration 20nmol/L handles donor and reconstructed embryo simultaneously, the influence to its developmental capacity；
1.9 data statistics
The data obtained is for statistical analysis by SPSS17.0 softwares, and it is same batch experiment that experiment, which is often repeated once,
It is tested under the ovary and experimental condition of identical source, all experiments are at least repeated 3 times.
2 interpretations of result
Influence of the 5-Aza-CdR processing reconstructed embryos of 2.1 various concentrations to HMC vitro Development of Embryos
Compare 40nmol/L, 20nmol/L, 10nmol/L, tetra- kinds of concentration 5-Aza-CdR processing of 5nmol/L, 0nmol/L
HMC reconstructed embryo 72h, the influence to its developmental capacity, the results showed that：5-Aza-CdR processing 72h can improve the division of reconstructed embryo
Rate, mulberry body rate, blastocyst rate and blastomere number, for each processing group, 20nmol/L processing group can significantly improve blastaea
Rate, 10nmol/L and 20nmol/L processing group can significantly improve blastomere number, and two group differences are not notable, refer to table
1, the 7th day blastaea developmental state is shown in that Fig. 1, Hoechst33342 dyeing blastomeres are shown in Fig. 2.
Influence of the 1 various concentration 5-Aza-CdR processing reconstructed embryos of table to HMC vitro Development of Embryos
Same column difference Superscript letters indicate significant difference (P<0.05).
2.2 5-Aza-CdR handle influence of the reconstructed embryo different time to HMC vitro Development of Embryos
Utilize 20nmol/L 5-Aza-CdR processing HMC reconstructed embryos different time (96h, 72h, 48h, for 24 hours, 0h), statistics
The embryonic development situation in each period finds that 5-Aza-CdR processing can effectively improve the division rate of reconstructed embryo, mulberry body rate, blastaea
Rate and blastomere number refer to table 2 and Fig. 3 wherein 72 hours processing groups can significantly improve blastocyst rate and blastomere number.
Influence of the table 220nmol/L 5-Aza-CdR processing reconstructed embryo different times to HMC vitro Development of Embryos
Same column difference Superscript letters indicate significant difference (P<0.05).
2.3 5-Aza-CdR handle the influence of donor and reconstructed embryo to HMC vitro Development of Embryos simultaneously
It is best dense with 0 μm of ol/L, 0.25 μm of ol/L, 0.5 μm of ol/L and 1 μm of ol/L, tetra- processing groups and reconstructed embryo respectively
Degree 20nmol/L handles donor and reconstructed embryo simultaneously, the results are shown in Table 3, each processing group reconstructed embryo developmental rate and blastaea total cell number phase
Control group is even reduced without significantly improving, when analysis may be due to handling donor and reconstructed embryo simultaneously, processing it is dense
Degree and time improper developed to late embryogenesis adversely affect, and refer to table 3, Fig. 4.
3 various concentration 5-Aza-CdR of table handles the influence of donor and reconstructed embryo to MHC vitro Development of Embryos simultaneously
Processing group (donor processing+reconstructed embryo processing)：1:0μmol/L+20nmol/L；2:0.25μmol/L+20nmol/L；
3:0.5μmol/L+20nmol/L；4:1μmol/L+20nmol/L.
Same column difference Superscript letters indicate significant difference (P<0.05).
The present invention after donor fibroblast progress 5-Aza-CdR processing 72h to having found, suitable concentration (0.25 μm of ol/L
~0.5 μm of ol/L) 5-Aza-CdR can reduce the fibroblastic methylation level of donor nuclei, and improve blastaea hair
Rate and blastaea total cell number are educated, as a result, it has been found that 20nmol/L, 72h are optimal treatment condition, the donor finally groped with front
After being handled simultaneously with the optimal treatment condition of embryo, it is found that there is no facilitation, analyses for development of such processing to embryo
It may be due to after handling in succession donor and reconstructed embryo, the medicament residue of 5-Aza-CdR itself and its cytotoxicity meeting
The effect of superposition is shown on reconstructed embryo.Reaching when therefore, to handle at the same time reduces DNA methylation level and raising embryo
The purpose that fetal hair is educated then needs to adjust respective concentration for the treatment of and time in experiment from now on, to reach optimum efficiency.It is suitable
The 5-Aza- of 5-Aza-CdR processing the donorcells 72h and 20nmol/L of suitable concentration (0.25 μm of ol/L~0.5 μm ol/L)
CdR processing reconstructed embryo 72h can reduce its DNA methylation level, and effectively improve the potentiality of development of the black pig HMC embryos in Debao.
The above is only the preferred embodiments of the present invention, and is not intended to limit the present invention in any form,
Every any simple modification made to the above embodiment according to the technical essence of the invention, equivalent variations and modification, belong to
In the range of technical solution of the present invention.
Claims (3)
1. a kind of drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao, which is characterized in that the black pig in Debao is manual
The drug of clone's reconstruct vitro Development of Embryos is 5-aza-2'-deoxycytidine；
0.25 μm of ol/L~0.5 μm ol/L of the 5-aza-2'-deoxycytidine concentration；Handle 72h.
2. the drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao as described in claim 1, which is characterized in that described
A concentration of 20nmol/L of 5-aza-2'-deoxycytidine handles 72h.
3. a kind of drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao as described in claim 1 is in the black pig cultivation in Debao
In application.
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Cited By (4)
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| CN113061572A (en) * | 2021-04-21 | 2021-07-02 | 广西壮族自治区畜牧研究所 | Micro-hole improvement method for culturing embryo without zona pellucida in manual cloning procedure |
| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN113215087A (en) * | 2021-05-31 | 2021-08-06 | 广西壮族自治区畜牧研究所 | Method for improving in-vitro maturation development rate of porcine oocytes by adopting agomelatine |
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
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Cited By (5)
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| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN113061572A (en) * | 2021-04-21 | 2021-07-02 | 广西壮族自治区畜牧研究所 | Micro-hole improvement method for culturing embryo without zona pellucida in manual cloning procedure |
| CN113215087A (en) * | 2021-05-31 | 2021-08-06 | 广西壮族自治区畜牧研究所 | Method for improving in-vitro maturation development rate of porcine oocytes by adopting agomelatine |
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
| CN114107180B (en) * | 2021-10-21 | 2024-03-08 | 湖北省农业科学院畜牧兽医研究所 | Zona pellucida-free somatic clone 1-cell stage embryo polymerization and in vitro culture method |
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