CN110016483A - A kind of method of cumulus cell gene function in research oocyte in vitro maturation - Google Patents
A kind of method of cumulus cell gene function in research oocyte in vitro maturation Download PDFInfo
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- 230000035800 maturation Effects 0.000 title claims abstract description 36
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 23
- 210000001771 cumulus cell Anatomy 0.000 title claims abstract description 22
- 238000000338 in vitro Methods 0.000 title claims abstract description 20
- 238000011160 research Methods 0.000 title claims abstract description 9
- 229920000089 Cyclic olefin copolymer Polymers 0.000 claims abstract description 25
- 241000700605 Viruses Species 0.000 claims abstract description 20
- 238000001890 transfection Methods 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 230000002611 ovarian Effects 0.000 claims abstract description 10
- 210000002394 ovarian follicle Anatomy 0.000 claims abstract description 6
- 239000002480 mineral oil Substances 0.000 claims abstract description 5
- 235000010446 mineral oil Nutrition 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000000520 microinjection Methods 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 8
- 210000000130 stem cell Anatomy 0.000 description 8
- 239000013642 negative control Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000004720 fertilization Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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Abstract
The invention discloses a kind of methods of cumulus cell gene function in research oocyte in vitro maturation, include the following steps: the preparation of mature dish: taking 20.83uL maturation liquid in 200uL centrifuge tube, and add 1uL slow virus, piping and druming mixes；Mature liquid after mixing is done into culture drop in culture dish, and 3mL mineral oil is added, culture dish is placed on incubator balance 3-5h；Slow-virus transfection COCs: COCs is cleaned three times from the COCs that picks out in ovarian follicle, then with maturation liquid IVM；Cleaned COCs is transfected 30-50h, the method that slow-virus transfection cumulus-oocyte complex of the invention carrys out ovarian cumulus gene function in invention oocyte maturation, the expression of target gene or albumen in interference cumulus cell is realized by slow-virus transfection, the problem of efficiently solving cumulus cell not and can be carried out microinjection, and the effect that ovarian cumulus gene plays in Oocyte Maturation Process can be played well.
Description
Technical field
The present invention relates to embryo engineering field, cumulus cell gene function in specifically a kind of research oocyte in vitro maturation
The method of energy.
Background technique
With the development and maturation of Embryo engineering technology, egg mother cell has realized maturation in vitro, in vitro fertilization, external at present
Culture and the production and culture of clone embryos, and the development of these technologies promotes Embryo engineering technology commercialization to be possibly realized.
But the egg mother cell quantity and quality of maturation in vitro cannot be guaranteed, to affect in vitro fertilization, clone embryos and embryo
The efficiency of development finally affects the application and popularization of Embryo engineering technology in production.Generally, the egg mother cell obtained in vitro
It is all as made of different layers of cumulus cells package, the package more Oocyte qualities of the number of plies are better.It is only good
Egg mother cell can just reach maturity and successful fertilization is to form embryo.Similarly, the cumulus cell of oocyte peripheral package
Important function has been played in Oocyte Maturation Process, it will do it information interchange between egg mother cell, it may be assumed that paracrine and
Gap connection, to promote oocyte maturation, fertilization and early embryonic development.
When going deep into the influence of some gene or albumen in invention cumulus cell to oocyte maturation, it is necessary to up-regulation or
The expression for handling the gene or protein level is lowered, so that whether the maturation for observing egg mother cell is affected, it is mainly mature
Whether rate change, some mRNA and whether protein level changes and whether relevant signal path receives shadow
It rings.Generally, such invention is all to realize gene or protein expression up-regulation by microinjection or lower.But it is wrapped in ovum
The cumulus cell quantity of mother cell periphery is relatively more, and cumulus cell cannot achieve microinjection different from egg mother cell, because
This cannot achieve the up-regulation or downward of gene or expressing quantity, to bring a degree of difficult and shadow to experiment invention
It rings.
Summary of the invention
The technical problem to be solved in the present invention is that providing cumulus cell gene in a kind of research oocyte in vitro maturation
The method of function, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme:
The method of cumulus cell gene function, includes the following steps: in a kind of research oocyte in vitro maturation
(1) preparation of mature dish
It takes 20.83uL oocyte in vitro maturation culture solution in 200uL centrifuge tube, adds 1uL slow virus, piping and druming is mixed
It is even；Mature liquid after mixing is done into culture drop in culture dish, and 3mL mineral oil is added, culture dish is placed on incubator balance
3-5h；
(2) slow-virus transfection cumulus-oocyte complex (COCs)
COCs is cleaned three times from the COCs that picks out in ovarian follicle, then with maturation liquid IVM；Cleaned COCs is transfected
30-50h, and fluorescence picture is clapped under fluorescence inverted microscope.
Preferably, in step (1), the temperature in incubator is 38.5 DEG C, CO2Volume fraction be 5%.
Preferably, in step (1), culture dish is placed on incubator balance 4h.
Preferably, in step (2), COCs be from diameter be 3-5mm ovarian follicle in select, select ovarian cumulus package the number of plies it is identical and
The COCs of uniform quality.
Preferably, in step (2), COCs transfection time is 42h.
Compared with prior art, the beneficial effects of the present invention are:
The present invention studies ovarian cumulus gene function in oocyte maturation by slow-virus transfection cumulus-oocyte complex
The method of energy realizes the expression of target gene or albumen in interference cumulus cell by slow-virus transfection, to efficiently solve
Cumulus cell not can be carried out the problem of microinjection, while can play ovarian cumulus gene well again in Oocyte Maturation Process
The effect of performance.The present invention is using slow-virus transfection cumulus-oocyte complex come ovarian cumulus base in invention oocyte maturation
Because the method for function has practical significance and utility value: on the one hand, from a cost perspective, being compared to microinjection, slow virus
Transfection only needs to consume certain reagent and material, does not need to put into a large amount of manpower, material resources and financial resources.On the other hand, it is grasped from experiment
It is easy to operate from the point of view of making, it is not limited by experiment condition, is operated at any time convenient for experimenter.
In conclusion the present invention is studied in oocyte maturation using slow-virus transfection cumulus-oocyte complex
The method of ovarian cumulus gene function can be widely used in the scientific invention in embryo engineering laboratory, to promote embryo's biology skill
Application of the art in animal husbandry and human reproduction's medicine.
Detailed description of the invention
Fig. 1 is enters dish picture before slow-virus transfection, and wherein a is blank control group-light field, and b is slow virus negative control group-
Light field, c are slow virus experimental group-light field；
Fig. 2 is enters dish picture after slow-virus transfection, and wherein 1a is blank control group-light field, 1b slow virus negative control group-
Light field, 1c slow virus experimental group-light field, 2a blank control group-fluorescence, 2b slow virus negative control group-fluorescence, 2c slow virus are real
Test group-fluorescence.
Specific embodiment
Material, reagent used in following embodiment and its source.
Material
The material that the present invention uses has: domestic blue electron gun head, yellow pipette tips, white pipette tips, 2.5 μ L (eppendorf of liquid-transfering gun
Research plus), 100uL (Dragon Lab, 10-100 μ L), 1mL (Dragon Lab, 100-1000 μ L), centrifuge tube
(200 μ L, Axygen), 35mm × 10mm ware (FALCON, 351008), marking pen, inverted fluorescence microscope (Olympus).
Reagent
The reagent that the present invention uses has: mature liquid IVM (in vitro maturation solution),
Lentivirus (Ji Ma company), mineral oil (Sigma, M8410).
The method of cumulus cell gene function, includes the following steps: in a kind of research oocyte in vitro maturation
1.1. the preparation of mature dish
1. taking 20.83uL maturation liquid in 3 200uL centrifuge tubes respectively, and negative labeled as blank control group, slow virus
Control group, slow virus experimental group；
2. being separately added into 1uL maturation liquid, 1uL negative control slow virus, 1uL experiment slow virus in 1., piping and druming is mixed；
3. doing culture drop in 35mm culture dish, and 3mL mineral oil is added；
4. carrying out group label on culture dish, and culture dish is placed on 38.5 DEG C of 5%CO2 incubator balance 4h.
1.2. slow-virus transfection cumulus-oocyte complex (COCs)
1. sorting out COCs of the diameter in 3-5mm ovarian follicle, selecting ovarian cumulus package, the number of plies is identical and the COCs of uniform quality；
2. being cleaned three times with the COCs that IVM liquid will be singled out；
3. the random simultaneously average mark of cleaned COCs in three groups；
4. transfecting 42h, and fluorescence picture is clapped under fluorescence inverted microscope.
1.3. slow-virus transfection COCs experimental result
As a result as depicted in figs. 1 and 2, this experiment is divided into three groups, blank control group, slow virus negative control group and slow
Viral experiment group.Wherein, blank control group is without any processing, and slow virus negative control group is added to containing only green glimmering
The Lentivirus of optical label, slow virus experimental group are added to for the shRNA of target gene and with green fluorescence mark
The slow virus of label.
It has been observed that the equal redgreen of the nucleus of blastaea trophocyte in blank control group under inverted fluorescence microscope
There is green fluorescence on the nucleus of blastaea trophocyte in fluorescence, slow virus negative control group and slow virus experimental group.This
Invention realizes that target gene is specific expressed in cumulus cell by slow-virus transfection COCs, can effectively realize target gene
It strikes low, and then can further investigate whether the target gene will affect diffusion and the subtrahend of ovarian cumulus in Oocyte Maturation Process
The process of division, may finally determine the target gene whether the important function played in Oocyte Maturation Process, together
When be also it is subsequent further investigation lay the foundation.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (5)
1. a kind of method of cumulus cell gene function in research oocyte in vitro maturation, which is characterized in that including walking as follows
It is rapid:
(1) preparation of mature dish
It takes 20.83uL oocyte in vitro maturation culture solution in 200uL centrifuge tube, adds 1uL slow virus, piping and druming mixes；
Mature liquid after mixing is done into culture drop in culture dish, and 3mL mineral oil is added, culture dish is placed on incubator balance 3-
5h；
(2) slow-virus transfection cumulus-oocyte complex (COCs)
COCs is cleaned three times from the COCs that picks out in ovarian follicle, then with maturation liquid IVM；Cleaned COCs is transfected 30-
50h, and fluorescence picture is clapped under fluorescence inverted microscope.
2. cumulus cell base during specificity invention cumulus oocyte complex maturation in vitro according to claim 1
Because of the method for function, which is characterized in that in step (1), the temperature in incubator is 38.5 DEG C, CO2Volume fraction be 5%.
3. cumulus cell base during specificity invention cumulus oocyte complex maturation in vitro according to claim 1
Because of the method for function, which is characterized in that in step (1), culture dish is placed on incubator balance 4h.
4. cumulus cell base during specificity invention cumulus oocyte complex maturation in vitro according to claim 1
Because of the method for function, which is characterized in that in step (2), COCs is to select ovarian cumulus package from diameter to select in 3-5mm ovarian follicle
The number of plies is identical and the COCs of uniform quality.
5. cumulus cell base during specificity invention cumulus oocyte complex maturation in vitro according to claim 1
Because of the method for function, which is characterized in that in step (2), COCs transfection time is 42h.
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|---|---|---|---|---|
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-
2019
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| CAO, LH等: "Expression patterns of ZO-1/2 and their effects on porcine oocyte in vitro maturation and early embryonic development", 《THERIOGENOLOGY》 * |
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