CN109820819A - A kind of improvement injection mixed liquor method of preparation and use - Google Patents
A kind of improvement injection mixed liquor method of preparation and use Download PDFInfo
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Abstract
The invention belongs to injection technical fields, disclose a kind of improvement injection mixed liquor method of preparation and use.The injection mixed liquor of improvement is in proportion by transfection liposome vectors, exogenous plasmid and external source miRNAs solution composition；Preparation method includes: the ddH by the exogenous plasmid of building using sterilizing2O diluted concentration is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O diluted concentration is 20pmoL/ μ L；It is ready for transfection liposome vectors；And according to transfection liposome vectors: Sox9a exogenous plasmid: 2~4 μ L of proportional arrangement mixed liquor of outer miRNAs (can be one or more mixtures)=2:1:1.Application method uses the external microinjection combination electrotransfection of sexual gland.The present invention can control the differentiation and the regeneration of adult fish sexual gland of 6 months zebra fish spermaries and ovary by adding different proportion and different types of external source miRNAs into original mixed liquor.
Description
Technical field
The invention belongs to injection technical fields more particularly to a kind of improvement to inject mixed liquor method of preparation and use.
Background technique
Currently, the prior art commonly used in the trade is such that
Between past 10 years, zebra fish has become a kind of mould with very high researching value as the carrier of kinds of experiments
Type, zebra fish more levels off to mammal and some mammals such as mankind and mouse in evolution has homologous gene.
Zebra fish shows certain plasticity during Sex Differentiation, while having very strong adjustment mechanism.
The development and Sex Differentiation of the adjustable vertebrate sexual gland of miRNAs and FSH/cAMP/MAPK/Sox9 signal path, however have
The regulated and control network of body is unintelligible, projects injection technique in vitro by a kind of zebra fish, can inject gene pairs zebra by external source
The Gonad Differentiation of fish is regulated and controled.
It is currently known and only will increase sperm in spermary to 6 months male zebra fish external source injection Sox9a foreign genes
Density reduces the quantity of sperm mother cell.This simple injecting method exist it is functional single, use scope limitation lack
Point.Promote the regenerated report of sexual gland hyperplasia, especially ovarian follicle few.
In conclusion problem of the existing technology is:
The foreign gene of per injection is single, is merely able to orient the differentiation situation of regulation zebra fish spermary to some direction.
When injecting single foreign gene, the degree of zebra fish spermary differentiation is low, and effect is not significant.
There are no fine methods to promote ovarian follicle newborn.
Solve the meaning of above-mentioned technical problem:
The present invention provides the technologies that one kind can control zebra fish gonad development and differentiation to a certain extent, after being
Fish or the scientific research of mammality Gonad Differentiation provide a kind of new idea and method.And adult fish reproduction is asked
Topic provides a new breakthrough mouth, while the problem infertile to the mankind also provides orientation treatment.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of improvement to inject mixed liquor method of preparation and use.
The invention is realized in this way mixed liquor is injected in a kind of improvement, the improvement injection mixed liquor is by transfection liposome
Carrier, exogenous plasmid and external source miRNAs solution composition；
Example transfects liposome vectors: Sox9b exogenous plasmid: external source miRNAs solution==2:1:1 in mass ratio；
The external source miRNAs solution is external source miR141, in external source miR-734, external source miR430a, external source miR218a
One or more mixtures.
Further, Sox9a+miR430a combination promotes sperm mother cell hyperplasia, and the exogenous plasmid of building is used sterilizing
ddH2O diluted concentration is 500ng/ μ L；The miRNAs of customization is used into the ddH of sterilizing again2O diluted concentration is 20pmoL/ μ L, is pressed
Transfect liposome vectors: Sox9a exogenous plasmid according to mass ratio: the proportional arrangement improvement injection of external source miR430a=2:1:1 is mixed
Close liquid.
Further, Sox9a+miR141+miR734 combination promotes class ovary to convert to spermary, and the exogenous plasmid of building is made
With the ddH of sterilizing2O diluted concentration is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O diluted concentration is
20pmoL/μL；Liposome vectors: Sox9a exogenous plasmid: external source miR141: external source miR-734=are transfected according to mass ratio
The proportional arrangement improvement injection mixed liquor of 4:2:1:1.
Further, Sox9b+miR218a combination promotes egg cell hyperplasia, and the exogenous plasmid of building is used sterilizing
ddH2O diluted concentration is 500ng/ μ L；The miRNAs of customization is used into the ddH of sterilizing again2O diluted concentration is 20pmoL/ μ L, is pressed
Liposome vectors: Sox9b exogenous plasmid: the proportional arrangement mixed liquor of external source miR218a=2:1:1 are transfected according to mass ratio.
It is an object of the present invention to provide a kind of preparation methods of improvement injection mixed liquor, comprising:
The exogenous plasmid of building is used the ddH of sterilizing by the first step2O dilution；
The miRNAs of customization is used the ddH of sterilizing by second step2O dilution；
Third step prepares transfection liposome vectors；
4th step, according to transfection liposome vectors: Sox9a/b exogenous plasmid: the proportional arrangement of external source miRNA=2:1:1
Mixed liquor；
5th step pinpoints microinjection, power-up transfection according to body surface projection.
Further, in the first step, the exogenous plasmid of building is used into the ddH of sterilizing2O is diluted to concentration: 500ng/ μ L.
Further, in second step, the miRNAs of customization is used into the ddH of sterilizing2O is diluted to concentration: 20pmoL/ μ L.
Another object of the present invention is to provide reasonably combined, the outfit mix preparations of exogenous plasmid and external source miRNAs
The differentiation of zebra fish sexual gland can be regulated and controled.
Further object of the invention is to provide the external medication administration method of improvement injection mixed liquor described in one kind, described
The external medication administration method of improvement injection mixed liquor includes: according to sexual gland body surface projection, by improvement injection mixed liquor using accurately
Sexual gland fixed-point injection and electroporation/electrotransfection increase tissue and cell medication effect.
In conclusion advantages of the present invention and good effect are as follows:
The present invention is obtained by above-mentioned experimental method during gonad development and differentiation are probed into zebra fish body
Obtained unexpected technical effect.It is well known that miRNAs can generate inhibitory effect, and external source base to the expression of target gene
The injection of cause then belongs to the overexpression of gene, and the present invention is by combining two methods, according to zebra generally acknowledged both at home and abroad
The signal path of fish Gonad Differentiation, carry out it is tentative probe into, by overexpression to different genes or inhibit to certain
Gonad development and the differentiation of zebra fish are effectively controlled in degree.
The method of zebra fish Gonad Differentiation research is mainly stayed at present: in embryonic stage, passing through different technologies means
The Individual genes of zebra fish knock out/drop or Enhanced expressing, to probe into Individual genes in zebra fish growth and development process
The effect of middle Gonad Differentiation.The injection of common foreign gene is carried out in vivo to zebra fish, to probe into foreign gene to spot
The one-side effect of horse fish Gonad Differentiation.
The present invention has filled up the blank of Gonad Differentiation method in domestic and international specific regulatory control zebra fish body, can be effectively special
The degree of Gonad Differentiation and direction in zebra fish body are controlled anisotropicly, are had to as the regenerated research of model animal zebra fish sexual gland
There is very important effect.
The present invention can promote sexual gland again by adding different proportion and different types of mixed liquor into original mixed liquor
Raw or Dot Blots Sex, different ingredient combined effects are different:
Sox9a+miR430a combination promotes sperm mother cell hyperplasia.The exogenous plasmid of building is used into the ddH of sterilizing2O is dilute
Releasing concentration is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O diluted concentration be 20pmoL/ μ L, according to turn
Contaminate liposome vectors: Sox9a exogenous plasmid: the proportional arrangement mixed liquor 1 of external source miR430a=2:1:1.
Sox9b+miR218a combination promotes ovarian follicle hyperplasia.The exogenous plasmid of building is used into the ddH of sterilizing2O dilution is dense
Degree is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O diluted concentration is 20pmoL/ μ L, according to transfection rouge
Plasmid vector: Sox9b exogenous plasmid: the proportional arrangement mixed liquor 4 of external source miR218a=:2:1:1.
Sox9a+miR141+miR734 combination promotes class ovary to convert to spermary, and the exogenous plasmid of building is used sterilizing
DdH2O diluted concentration is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O diluted concentration is 20pmoL/ μ
L；According to transfection liposome vectors: Sox9a exogenous plasmid: external source miR141: the proportional arrangement of external source miR-734=4:2:1:1
Mixed liquor 2.
It is another object of the present invention to improve medication administration method, i.e., according to body surface projection, pass through the micro- note of external sexual gland
Penetrate+electroporation/electrotransfection.
The external medication administration method of improvement injection mixed liquor provided by the invention can accurately pinpoint dispensing, reduce waste with it is non-
Special medication side effect；Turn to help drug into intracellular, increase medication effect in addition, auxiliary is electric.
Detailed description of the invention
Fig. 1 is improvement injection mixed liquor preparation method flow chart provided in an embodiment of the present invention.
Fig. 2 is simple injection Sox9a plasmid spermary Histological section figure provided in an embodiment of the present invention.
Fig. 3 is action diagram of the injection mixed liquor provided in an embodiment of the present invention to Gonad Differentiation in June.
Fig. 4 is that injection mixed liquor provided in an embodiment of the present invention promotes 1.5 years old adult fish sexual gland regeneration figure.
Fig. 5 is the statistical analysis figure of Fig. 2-4 provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In the prior art, the foreign gene of per injection is single, is merely able to orient regulation zebra fish spermary to some direction
Differentiation situation；Use scope has limitation.
In order to solve the above technical problems, below with reference to concrete scheme, the present invention will be described in detail.
Improvement provided in an embodiment of the present invention injection mixed liquor include: transfection liposome vectors, Sox9a/b exogenous plasmid,
External source miRNAs solution.
Wherein, liposome vectors: Sox9a/b exogenous plasmid: external source miRNAs (can be one or more mixtures) are transfected
=2:1:1.
In embodiments of the present invention, external source miRNAs include: external source miR141, it is external source miR-734, external source miR430a, outer
One of source miR218 or a variety of mixtures.
In embodiments of the present invention, Sox9a+miR430a combination promotes sperm mother cell hyperplasia, and the exogenous plasmid of building is made
With the ddH of sterilizing2O diluted concentration is 500ng/ μ L；The miRNAs of customization is used into the ddH of sterilizing again2O diluted concentration is
20pmoL/ μ L, transfect liposome vectors according to mass ratio: Sox9a exogenous plasmid: the ratio of external source miR430a=2:1:1 is matched
Set improvement injection mixed liquor.
In embodiments of the present invention, Sox9a+miR141+miR734 combination promotes class ovary to convert to spermary, by building
Exogenous plasmid uses the ddH to sterilize2O diluted concentration is 500ng/ μ L；Secondly the miRNAs of customization is used into the ddH of sterilizing2O is dilute
Releasing concentration is 20pmoL/ μ L；Liposome vectors: Sox9a exogenous plasmid: external source miR141: external source are transfected according to mass ratio
The proportional arrangement improvement injection mixed liquor of miR-734=4:2:1:1.
In embodiments of the present invention, Sox9b+miR218a combination promotes ovarian follicle hyperplasia, and the exogenous plasmid use of building is gone out
The ddH of bacterium2O diluted concentration is 500ng/ μ L；The miRNAs of customization is used into the ddH of sterilizing again2O diluted concentration is 20pmoL/ μ
L transfects liposome vectors: Sox9b exogenous plasmid: the proportional arrangement mixed liquor of external source miR218=2:1:1 according to mass ratio.
As shown in Figure 1, improvement injection mixed liquor configuration method provided in an embodiment of the present invention includes:
The exogenous plasmid of building is used the ddH of sterilizing by S1012O is diluted to following concentration: 500ng/ μ L.
The miRNAs of customization is used the ddH of sterilizing by S1022O is diluted to following concentration: 20pmoL/ μ L.
S103 prepares transfection liposome vectors.
S104, according to transfection liposome vectors: Sox9a/b exogenous plasmid: outer miRNAs (can be one or more mixing
Object)=2:1:1 2~4 μ L of proportional arrangement mixed liquor.
S105 pinpoints microinjection, power-up transfection according to body surface projection.
The invention will be further described combined with specific embodiments below.
Embodiment:
1), zebra fish prepares
(1) used drainage that MS222 (anesthetic) is diluted to 1x.
(2) zebra fish for preparing 5 months and 18 months, anaesthetizes zebra fish using the MS222 solution of 1x.
(3) after zebra fish inaction ability, zebra fish is put under microscope using cystosepiment with groove fixation.
2), the external sexual gland positioning of zebra fish:
The body surface projection position of injection site should be located at pectoral fin upper end horizontal line and abdomeinal fin front end vertical line infall is (red
Color dotted line) the top place about 1mm, external projection microinjection inserting needle position is as shown in Figure 2.
3), injection:
The mixed liquor for the different proportion that injection is configured according to configuration method provided in an embodiment of the present invention and existing
Sox9a/b foreign gene injection.
(1) mixed liquor 1: according to transfection liposome vectors: Sox9a exogenous plasmid: the ratio of external source miR430a=2:1:1
It configures mixed liquor 1 (such as Fig. 4 a, 4d).
(2) mixed liquor 2: according to transfection liposome vectors: Sox9a exogenous plasmid: external source miR141: external source miR-734=
The proportional arrangement mixed liquor 2 (such as Fig. 4 b, 4e) of 4:2:1:1.
(3) mixed liquor 3: according to transfection liposome vectors: Sox9b exogenous plasmid: the ratio of external source miR430a=2:1:1
Configure mixed liquor 3.
(4) mixed liquor 4: according to transfection liposome vectors: Sox9b exogenous plasmid: the ratio of external source miR218=:2:1:1
It configures mixed liquor 4 (such as Fig. 4 c, 4f).
(5) existing injection: Sox9a foreign gene (such as Fig. 2).
4), injecting method:
(1) dopey zebra fish is used, sexual gland position is substantially determined according to ideograph, the squama at the position is taken out with tweezers
Piece.
(2) osculum is scraped off with scissors or pocket knife, find sexual gland, be careful not to firmly excessive in order to avoid squeezing internal organ influence
Survival after zebra fish injection.
(3) syringe needle is penetrated into sexual gland, steps on pedal injection mixed liquor 2ul.The too deep of syringe needle bundle is careful not in order to avoid puncturing
Air bladder causes zebra fish dead.
(4) in order to improve plasmid/miRNA into histiocytic efficiency, the positive and negative anodes of electroporation tungsten needle are placed on respectively
The front of injection position at back, starts electroporation, it is as follows that electricity turns parameter:
Voltage: 50V.
Burst length: 30ms.
Interpulse period: 5s.
Pulse number: 4 times.
5) it, detects
5 months zebra fish are injected respectively using mixed liquor 1, mixed liquor 2, mixed liquor 4, use mixed liquor 3, mixing
Liquid 4 injects 18 months zebra fish respectively.
Zebra fish is dissected after waiting 3~4 weeks to be injected per progress one injection in each 3 days, carries out experimental verification.
Fig. 2 (simple injection Sox9a plasmid spermary Histological section figure) is injection mixed liquor 1 provided in an embodiment of the present invention
With injection 2 result of mixed liquor.
The influence that testis occurs for external source Sox9a/Sox9b coupling specificities miRNA and ovarian follicle occurs.The following are experiment numbers
According to:
As shown in Fig. 3 (action diagram of the injection mixed liquor to Gonad Differentiation in June), the testis of mature zebra fish occurs and ovarian follicle
The histological observation of generation.Based on cellular morphology, germ line cell is divided into three classes: germline stem cell (Gc), mitosis concentration dye
Chromaticness nucleus (Cn) and maturity gamete (Gm).Gc includes spermatogonium, oogonium or other undifferentiated primordial germs
Cell.It (Cn) include sperm mother cell or primary egg mother cell (poc), gamete includes spermatid or sperm (Sc), and ovarian follicle includes just
Five stages (I-V) of grade ovarian follicle and secondary follicle.The egg mother cell (dO) of degeneration indicated with arrows.Some widened reproductions are thin
Born of the same parents' (indicated by an arrow) look like the core week egg mother cell (or the reproduction cell for being considered as denaturation) of early stage.
As shown in Fig. 4 (injection mixed liquor promotes 1.5 years old adult fish sexual gland regeneration figure), the old fish of histological observation (1.5 years old)
Testis occurs and ovarian follicle occurs.A-c shows testis；D-f shows each stage that ovarian follicle occurs: stage Ia, Ib, II, III
With IV egg mother cell.
Shown in Fig. 5 (the statistical analysis figure of Fig. 2-4), in spermatogenesis and oogenetic each stage to reproduction cell
Cell count statistical analysis.The ratio for comparing each classification, 5mf fish (a, b) and 1-1.5 years old fish (c) difference it is special
Property miRNA-Sox9a/b processing in.* (p < 0.05), * * (p < 0.01).Scale bar=50 μm (testis) and 500 μm (ovary).
Below with reference to interpretation of result, the invention will be further described.
To the Gonad Differentiation regulating and controlling effect of mature zebra fish (6 months):
Sox9a+miR-430a: chromosome concentrating cells (cn) and undifferentiated spermatogonium (Gc) in spermary be can be improved
Ratio promotes the degradation of first oocyte in ovary.
Sox9a+miR-141+miR-734: can be improved the ratio of spermatoblast, and induction ovary is converted to spermary.
Sox9b+miR-218a: the development of each stage ovarian follicle can be promoted, but reduce sperm number, improve the ratio of ovarian follicle
Example.
To the Gonad Differentiation regulating and controlling effect of old zebra fish (18 months):
Sox9a+miR-430a: the ratio of Gc and cn is improved.
Sox9b+miR-218a: it is capable of the ratio of newborn ovarian follicle.
Sox9b+miR430a: the development of first oocyte is improved in part.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. mixed liquor is injected in a kind of improvement, which is characterized in that the improvement injection mixed liquor is by transfection liposome vectors, external source matter
Grain and external source miRNAs solution composition；
Example transfects liposome vectors: Sox9a/b exogenous plasmid: external source miRNAs solution==2:1:1 in mass ratio；
The external source miRNAs solution is external source miR141, external source miR-734, external source miR430a, one in external source miR218a
Kind or a variety of mixtures.
2. improvement injection mixed liquor as described in claim 1, which is characterized in that Sox9a+miR430a combination promotes essence female thin
The improvement injection mixed liquor of born of the same parents' hyperplasia transfects liposome vectors: Sox9a exogenous plasmid: external source miR430a=according to mass ratio
The proportional arrangement of 2:1:1.
3. improvement injection mixed liquor as described in claim 1, which is characterized in that Sox9a+miR141+miR734 combination promotes
The improvement injection mixed liquor that class ovary is converted to spermary transfects liposome vectors: Sox9a exogenous plasmid: external source according to mass ratio
MiR141: the proportional arrangement of external source miR-734=4:2:1:1.
4. improvement injection mixed liquor as described in claim 1, which is characterized in that
Sox9b+miR218a combination promotes the improvement injection mixed liquor of primary follicle hyperplasia to carry according to mass ratio transfection liposome
Body: Sox9b exogenous plasmid: the proportional arrangement of external source miR218=2:1:1.
5. a kind of preparation and application of improvement injection mixed liquor as described in claim 1, method include:
The exogenous plasmid of building is used the ddH of sterilizing by the first step2O dilution；
The miRNAs of customization is used the ddH of sterilizing by second step2O dilution；
Third step prepares transfection liposome vectors；
4th step, according to transfection liposome vectors: Sox9a/b exogenous plasmid: the proportional arrangement of external source miRNA=2:1:1 mixes
Liquid；
5th step pinpoints microinjection, power-up transfection according to body surface projection.
6. the preparation method of improvement injection mixed liquor as claimed in claim 5, which is characterized in that in the first step, by building
Exogenous plasmid uses the ddH to sterilize2O is diluted to concentration: 500ng/ μ L.
7. the preparation method of improvement injection mixed liquor as claimed in claim 5, which is characterized in that in second step, will customize
MiRNAs using sterilizing ddH2O is diluted to concentration: 20pmoL/ μ L.
8. a kind of mixing using the differentiation situation of the regulation zebra fish sexual gland of improvement injection mixed liquor preparation described in claim 1
Preparation.
9. a kind of external medication administration method of improvement injection mixed liquor as described in claim 1, which is characterized in that the improvement note
The external medication administration method for penetrating mixed liquor includes: that improvement injection mixed liquor is used sexual gland fixed-point injection according to sexual gland body surface projection
With electroporation/electrotransfection.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016076929A1 (en) * | 2014-11-13 | 2016-05-19 | University Of Iowa Research Foundation | Methods to generate epithelial cells |
| CN107868834A (en) * | 2017-12-04 | 2018-04-03 | 四川省农业科学院水产研究所 | The real-time quantitative PCR detection primer group and method of a kind of acipenser dabryanus sexual gland difference expression gene |
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2019
- 2019-02-22 CN CN201910133030.2A patent/CN109820819B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016076929A1 (en) * | 2014-11-13 | 2016-05-19 | University Of Iowa Research Foundation | Methods to generate epithelial cells |
| CN107868834A (en) * | 2017-12-04 | 2018-04-03 | 四川省农业科学院水产研究所 | The real-time quantitative PCR detection primer group and method of a kind of acipenser dabryanus sexual gland difference expression gene |
Non-Patent Citations (4)
| Title |
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| FRANCISCA M. REAL等: "A MicroRNA (mmu-miR-124) Prevents Sox9 Expression in Developing Mouse Ovarian Cells", 《BIOLOGY OF REPRODUCTION》 * |
| JING JING等: "Sex-Biased miRNAs in Gonad and Their Potential Roles for Testis Development in Yellow Catfish", 《PLOS ONE》 * |
| 杜新路等: "一种通过体表注射斑马鱼性腺的简易投药方法", 《上海海洋大学学报》 * |
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