CN109820819B - Preparation and application method of improved injection mixed solution - Google Patents
Preparation and application method of improved injection mixed solution Download PDFInfo
- Publication number
- CN109820819B CN109820819B CN201910133030.2A CN201910133030A CN109820819B CN 109820819 B CN109820819 B CN 109820819B CN 201910133030 A CN201910133030 A CN 201910133030A CN 109820819 B CN109820819 B CN 109820819B
- Authority
- CN
- China
- Prior art keywords
- exogenous
- mixed solution
- plasmid
- sox9a
- mirnas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of injection and discloses a preparation method and a use method of an improved injection mixed solution. The improved injection mixed solution is proportionally composed of a transfection liposome carrier, exogenous plasmids and exogenous miRNAs solution; the preparation method comprises the following steps: the constructed foreign plasmid was used with sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2The O dilution concentration was 20 pmoL/. mu.L; then preparing a transfection liposome carrier; and following transfection of the liposome vector: sox9a exogenous plasmid: 2-4 mul of mixed solution is prepared by mixing exogenous miRNA (one or more than one mixture) in a ratio of 2:1: 1. The application method adopts gonad in-vitro microinjection combined with electrotransfection. According to the invention, exogenous miRNAs with different proportions and different types are added into the original mixed solution, so that the differentiation of the spermary and ovary of the zebra fish and the gonad regeneration of adult fish can be controlled for 6 months.
Description
Technical Field
The invention belongs to the technical field of injection, and particularly relates to a preparation method and a use method of an improved injection mixed solution.
Background
Currently, the current state of the art commonly used in the industry is such that:
over the past decade, zebrafish have become a model of great research value as a vehicle for a variety of experiments, and zebrafish have evolved to more closely resemble mammals, and some mammals such as humans and mice have homologous genes.
The zebra fish exhibits certain plasticity in the sex differentiation process and has a strong regulation mechanism. miRNAs and FSH/cAMP/MAPK/Sox9 signal channels can regulate the development and sex differentiation of gonads of vertebrates, however, the specific regulation network is unclear, and the gonad differentiation of zebra fish can be regulated by externally injecting genes through an in vitro projection injection technology of the zebra fish.
It is known that exogenous injection of Sox9a gene into male zebra fish at 6 months can only increase the density of sperms in the spermary and reduce the number of spermatocytes. The simple injection method has the defects of single function and limited use range. There are few reports on the promotion of gonadal hyperplasia, particularly follicular regeneration.
In summary, the problems of the prior art are as follows:
the exogenous gene injected each time is single, and can only directionally regulate and control the differentiation condition of the testis of the zebra fish in a certain direction.
When a single exogenous gene is injected, the differentiation degree of the testis of the zebra fish is low, and the effect is not obvious.
There is no good way to promote follicle neogenesis.
The significance of solving the technical problems is as follows:
the invention provides a technology capable of controlling the development and differentiation of the sexual gland of the zebra fish to a certain extent, and provides a new thought and method for the later scientific research on the gonad differentiation of the fish or the mammal. And provides a new breakthrough for the adult fish breeding problem and provides a treatment direction for the human infertility problem.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method and a use method of an improved injection mixed solution.
The present invention is achieved by an improved injection mixture consisting of a transfection liposome carrier, exogenous plasmid and exogenous miRNAs solution;
transfecting a liposome vector according to a mass ratio: sox9b exogenous plasmid: exogenous miRNAs solution 2:1: 1;
the exogenous miRNAs solution is one or a mixture of exogenous miR141, exogenous miR-734, exogenous miR430a and exogenous miR218 a.
Further, Sox9a + miR430a combination promotes spermatocyte proliferation, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; the customized miRNAs were then applied to sterilized ddH2O dilution concentration 20 pmoL/. mu.l, transfection of liposome vector by mass ratio: sox9a exogenous plasmid: the modified injection mixed solution is prepared according to the ratio of exogenous miR430a to 2:1: 1.
Further, Sox9a + miR141+ miR734 combination promotes transformation of ovary-like to testis, and the constructed exogenous plasmid is sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2The O dilution concentration was 20 pmoL/. mu.L; transfecting a liposome vector according to the mass ratio: sox9a exogenous plasmid: exogenous miR 141: the modified injection mixed solution is prepared according to the ratio of exogenous miR-734 to 4:2:1: 1.
Further, Sox9b + miR218a combination promotes egg cell proliferation, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; the customized miRNAs were then applied to sterilized ddH2O dilution concentration 20 pmoL/. mu.l, transfection of liposome vector by mass ratio: sox9b exogenous plasmid: the mixture was prepared at a ratio of 2:1:1 with exogenous miR218 a.
One object of the present invention is to provide a method for preparing an improved injection mixture, comprising:
in the first step, the constructed foreign plasmid was used with sterilized ddH2Diluting with oxygen;
second, custom miRNAs were applied using sterilized ddH2Diluting with oxygen;
thirdly, preparing a transfection liposome carrier;
step four, according to the transfection liposome carrier: sox9a/b foreign plasmid: preparing a mixed solution according to the proportion of 2:1:1 of exogenous miRNA;
fifthly, carrying out fixed-point microinjection according to the body surface projection, and electrifying for transfection.
Further, in the first step, the constructed foreign plasmid was used with sterilized ddH2Dilution to concentration of O: 500 ng/. mu.L.
Further, in a second step, the tailored miRNAs are applied using sterilized ddH2Dilution to concentration of O: 20 pmoL/. mu.L.
Another object of the present invention is to provide a rational combination of exogenous plasmids and exogenous miRNAs, which is prepared with a mixed preparation capable of regulating and controlling the differentiation of gonads of zebra fish.
Still another object of the present invention is to provide a method for in vitro administration of the improved injectable solution, comprising: according to the gonad body surface projection, the improved injection mixed solution adopts accurate gonad fixed-point injection and electroporation/electrotransfection to increase the medication effect of tissues and cells.
In summary, the advantages and positive effects of the invention are:
in the process of exploring gonad development and differentiation in zebra fish bodies, unexpected technical effects are obtained through the experimental method. The invention combines two methods, tries to explore according to a signal path of zebra fish gonad differentiation recognized at home and abroad, and effectively controls the gonad development and differentiation of zebra fish to a certain extent by over-expression or inhibition of different genes.
The current method for researching the differentiation of the zebra fish gonads mainly comprises the following steps: in the embryonic period, individual genes of the zebra fish are knocked out/reduced or enhanced to express through different technical means, so that the gonad differentiation effect of the individual genes in the growth and development process of the zebra fish is explored. Common exogenous genes are injected into the body of the zebra fish, so that the unilateral effect of the exogenous genes on the differentiation of the zebra fish gonad is explored.
The method fills the blank of a method for specifically regulating and controlling the gonad differentiation in the zebra fish at home and abroad, can effectively and specifically control the degree and direction of the gonad differentiation in the zebra fish, and has very important effect on the research of the gonad regeneration of the zebra fish as a model animal.
The gonad regeneration or sex conversion can be promoted by adding different proportions and different types of mixed liquid into the original mixed liquid, and the combination effect of different components is different:
the Sox9a + miR430a combination promotes spermatocyte proliferation. The constructed foreign plasmid was used with sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2O dilution concentration was 20pmoL/μ L, according to the transfection liposome vector: sox9a exogenous plasmid: mixture 1 was prepared at a ratio of 2:1:1 of exogenous miR430 a.
The Sox9b + miR218a combination promotes follicular proliferation. The constructed foreign plasmid was used with sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2O dilution concentration was 20pmoL/μ L, according to the transfection liposome vector: sox9b exogenous plasmid: mixed solution 4 was prepared at a ratio of exogenous miR218 a: 2:1: 1.
Sox9a + miR141+ miR734 combination promotes transformation of ovary-like to testis, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2The O dilution concentration was 20 pmoL/. mu.L; following transfection of the liposome vector: sox9a exogenous plasmid: exogenous miR 141: mixed solution 2 was prepared at a ratio of exogenous miR-734 to 4:2:1: 1.
Another object of the invention is to improve the method of administration, i.e. by in vitro gonadal microinjection + electroporation/electrotransfection, according to the projection on the body surface.
The in-vitro administration method of the improved injection mixed solution can accurately perform fixed-point administration, and reduce waste and non-specific side effects; in addition, the auxiliary electrotransfer helps the medicine to enter the cells, and the medicine application effect is improved.
Drawings
FIG. 1 is a flow chart of a method for preparing an improved injection mixture according to an embodiment of the present invention.
FIG. 2 is a histological section of testis from a plasmid injected with Sox9a alone according to an embodiment of the present invention.
FIG. 3 is a graph showing the effect of the injection mixture on the differentiation of gonads in 6 months according to the embodiment of the present invention.
Fig. 4 is a graph of the injection mixture provided by the embodiment of the invention for promoting gonad regeneration of adult fish of 1.5 years old.
Fig. 5 is a graph of the statistical analysis of fig. 2-4 provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the prior art, the exogenous gene injected each time is single, and the differentiation condition of the testis of the zebra fish can be directionally regulated and controlled only in a certain direction; the application range is limited.
In order to solve the above technical problems, the present invention will be described in detail with reference to specific embodiments.
The embodiment of the invention provides an improved injection mixed solution, which comprises the following components: transfection liposome carrier, Sox9a/b exogenous plasmid, exogenous miRNAs solution.
Wherein, the transfection liposome carrier: sox9a/b foreign plasmid: exogenous miRNAs (which may be one or a mixture of more) 2:1: 1.
In embodiments of the invention, the exogenous miRNAs comprise: one or more mixtures of exogenous miR141, exogenous miR-734, exogenous miR430a and exogenous miR 218.
In the embodiment of the invention, the Sox9a + miR430a combination promotes spermatocyte proliferation, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; the customized miRNAs were then applied to sterilized ddH2O dilution concentration 20 pmoL/. mu.l, transfection of liposome vector by mass ratio: sox9a exogenous plasmid: the modified injection mixed solution is prepared according to the ratio of exogenous miR430a to 2:1: 1.
In the embodiment of the invention, the combination of Sox9a + miR141+ miR734 promotes the transformation from ovary-like to testis, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; second, the tailored miRNAs were applied using sterilized ddH2The O dilution concentration was 20 pmoL/. mu.L; transfecting a liposome vector according to the mass ratio: sox9a exogenous plasmid: exogenous miR 141: the modified injection mixed solution is prepared according to the ratio of exogenous miR-734 to 4:2:1: 1.
In the embodiment of the invention, the Sox9b + miR218a combination promotes follicular proliferation, and the constructed exogenous plasmid uses sterilized ddH2The O dilution concentration is 500 ng/mu L; the customized miRNAs were then applied to sterilized ddH2O dilution concentration 20 pmoL/. mu.l, transfection of liposome vector by mass ratio: sox9b exogenous plasmid: the mixture was prepared at a ratio of exogenous miR218 to 2:1: 1.
As shown in fig. 1, the method for preparing an improved injection mixture according to an embodiment of the present invention includes:
s101, constructing exogenous plasmid by using sterilized ddH2O was diluted to the following concentrations: 500 ng/. mu.L.
S102, Using sterilized ddH with custom miRNAs2O was diluted to the following concentrations: 20 pmoL/. mu.L.
S103, preparing a transfection liposome carrier.
S104, following transfection of the liposome vector: sox9a/b foreign plasmid: 2-4 mul of mixed solution is prepared by mixing exogenous miRNA (one or more than one mixture) in a ratio of 2:1: 1.
S105, performing fixed-point microinjection according to the body surface projection, and performing electric transfection.
The invention is further described with reference to specific examples.
Example (b):
1) preparation of zebra fish
(1) MS222 (anesthetic) was diluted to 1x with filtered water.
(2) Zebrafish were prepared for 5 months and 18 months and were anesthetized with 1x MS222 solution.
(3) After the zebra fish is disabled, the zebra fish is fixed under a microscope by using a foam board with grooves.
2) And locating the external gonad of the zebra fish:
the body surface projection position of the injection site should be located about 1mm above the intersection of the horizontal line at the upper end of the pectoral fin and the vertical line at the front end of the ventral fin (red dotted line), and the in vitro projection microinjection needle insertion position is shown in fig. 2.
3) And injection:
the injection is mixed solution with different proportions and the existing Sox9a/b exogenous gene injection prepared according to the preparation method provided by the embodiment of the invention.
(1) Mixed solution 1: following transfection of the liposome vector: sox9a exogenous plasmid: mixture 1 (see fig. 4a and 4d) was prepared at a ratio of 2:1:1 with exogenous miR430 a.
(2) And (2) mixed solution: following transfection of the liposome vector: sox9a exogenous plasmid: exogenous miR 141: mixed solution 2 was prepared at a ratio of exogenous miR-734 to 4:2:1:1 (see fig. 4b and 4 e).
(3) And (3) mixed solution: following transfection of the liposome vector: sox9b exogenous plasmid: mixture 3 was prepared at a ratio of 2:1:1 of exogenous miR430 a.
(4) And (4) mixed solution: following transfection of the liposome vector: sox9b exogenous plasmid: mixture 4 was prepared at a ratio of exogenous miR 218: 2:1:1 (see fig. 4c and 4 f).
(5) The existing injection comprises the following components: sox9a exogenous gene (see FIG. 2).
4) And an injection method:
(1) using an anesthetized zebrafish, the gonad position was roughly determined from the pattern chart, and the scale was removed from the site with forceps.
(2) And (4) scraping a small opening with a scissors or a knife to find the gonad, and paying attention not to press the internal organs too much to influence the survival of the zebra fish after injection.
(3) The needle is inserted into the gonad, and the pedal is stepped down to inject 2ul of the mixed liquid. Care should be taken not to prick too deeply with the needle to avoid piercing swim bladder and leading to death of zebra fish.
(4) In order to improve the efficiency of the plasmid/miRNA entering the tissue cells, the positive electrode and the negative electrode of a tungsten needle of an electrotransformation instrument are respectively placed on the front side and the back side of an injection position, the electrotransformation instrument is started, and electrotransformation parameters are as follows:
voltage: 50V.
Pulse time: 30 ms.
Pulse interval time: and 5 s.
The pulse times are as follows: 4 times.
5) And detecting
The zebra fish of 5 months old was injected with the mixed solution 1, the mixed solution 2, and the mixed solution 4, and the zebra fish of 18 months old was injected with the mixed solution 3 and the mixed solution 4, respectively.
Injecting every 3 days, and dissecting zebra fish after waiting for 3-4 weeks of injection, and carrying out experimental verification.
FIG. 2 (histological section of plasmid testis from Sox9a by simple injection) shows the results of injection mixture 1 and injection mixture 2 provided in the example of the present invention.
Influence of exogenous Sox9a/Sox9b coupled specific miRNAs on orchidogenesis and folliculogenesis. The following are experimental data:
histological observation of testicular and follicular development in mature zebrafish is shown in figure 3 (graph of effect of injected cocktail on gonad differentiation in month 6). Based on cell morphology, germ line cells are divided into three categories: germ stem cells (Gc), mitotically condensed chromatin nuclei cells (Cn) and mature gametes (Gm). Gc includes spermatogonium, oogonium or other undifferentiated primordial germ cells. (Cn) comprises spermatocytes or primary oocytes (poc), gametes comprise sperm cells or spermatozoa (Sc), and follicles comprise five stages (I-V) of primary follicles and secondary follicles. Degenerated oocytes (dO) are indicated by arrows. Some enlarged germ cells (indicated by arrows) look like early perinuclear oocytes (or are considered as degenerated germ cells).
As shown in fig. 4 (graph of the mixture injection for promoting gonad regeneration of adult fish of 1.5 years old), the old fish (1.5 years old) was histologically observed for testicular genesis and folliculogenesis. a-c indicates orchidogenesis; d-f show the various stages of folliculogenesis: stage Ia, Ib, II, III and IV oocytes.
FIG. 5 (statistical analysis of FIGS. 2-4) shows a statistical analysis of germ cell counts at various stages of spermatogenesis and oogenesis. The ratio of each class was compared in different specific miRNA-Sox9a/b treatments in 5mf fish (a, b) and 1-1.5 year old fish (c). (p < 0.05). or (p < 0.01). Scale bar 50 μm (testis) and 500 μm (ovary).
The invention is further described below in connection with the analysis of the results.
Gonad differentiation regulation effect on mature zebra fish (6 months):
sox9a + miR-430 a: can increase the ratio of chromosome condensation cells (cn) to undifferentiated spermatogonia (Gc) in the testis, and promote the degradation of primary oocytes in the ovary.
Sox9a + miR-141+ miR-734: can increase the proportion of spermatids and induce the transformation of ovary to testis.
Sox9b + miR-218 a: can promote the development of follicle at each stage, but reduce the number of sperms and increase the ratio of follicles.
Gonad differentiation regulation and control effect on the zebra fish (18 months):
sox9a + miR-430 a: the ratio of Gc to cn is increased.
Sox9b + miR-218 a: the proportion of newly born follicles.
Sox9b + miR430 a: partially enhance the development of primary oocytes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (5)
1. An improved injection mixture, wherein the improved injection mixture comprises a transfection liposome carrier, an exogenous plasmid and an exogenous miRNAs solution;
transfecting a liposome vector according to a mass ratio: sox9a/b foreign plasmid: exogenous miRNAs solution = =2:1: 1;
the exogenous miRNAs solution is one or a mixture of exogenous miR141, exogenous miR-734, exogenous miR430a and exogenous miR218 a.
2. The modified injection mixture of claim 1, wherein the Sox9a + miR430a in combination with the modified injection mixture for promoting spermatocyte proliferation transfects the liposome carrier in a mass ratio of: sox9a exogenous plasmid: exogenous miR430a =2:1:1 ratio configuration.
3. The modified injection mixture of claim 1, wherein Sox9a + miR141+ miR734, in combination with the modified injection mixture that promotes transformation of ovarian-like to testis, transfects the liposomal vector in a mass ratio of: sox9a exogenous plasmid: exogenous miR 141: exogenous miR-734=4:2:1:1 ratio configuration.
4. The modified injectable mixture of claim 1,
the modified injection mixed solution for promoting primary follicular proliferation by combining Sox9b + miR218a is transfected with a liposome carrier according to the mass ratio: sox9b exogenous plasmid: exogenous miR218=2:1:1 ratio configuration.
5. A mixed preparation for regulating and controlling the differentiation condition of the gonads of the zebra fish, which is prepared by using the improved injection mixed solution as claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910133030.2A CN109820819B (en) | 2019-02-22 | 2019-02-22 | Preparation and application method of improved injection mixed solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910133030.2A CN109820819B (en) | 2019-02-22 | 2019-02-22 | Preparation and application method of improved injection mixed solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109820819A CN109820819A (en) | 2019-05-31 |
| CN109820819B true CN109820819B (en) | 2021-03-23 |
Family
ID=66864188
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910133030.2A Expired - Fee Related CN109820819B (en) | 2019-02-22 | 2019-02-22 | Preparation and application method of improved injection mixed solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109820819B (en) |
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 |
-
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 |
|---|
| A MicroRNA (mmu-miR-124) Prevents Sox9 Expression in Developing Mouse Ovarian Cells;Francisca M. Real等;《BIOLOGY OF REPRODUCTION》;20130814;第89卷(第4期);第1-11页 * |
| Sex-Biased miRNAs in Gonad and Their Potential Roles for Testis Development in Yellow Catfish;Jing Jing等;《PLOS ONE》;20140917;第9卷(第9期);第1-9页 * |
| 一种通过体表注射斑马鱼性腺的简易投药方法;杜新路等;《上海海洋大学学报》;20180131;第27卷(第1期);第24-28页 * |
| 两个Sox9 基因在斑马鱼胚胎发育和成体性腺中的动态表达特征;梁清仪等;《山东农业科学》;20141231;第46卷(第7期);第20-24页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109820819A (en) | 2019-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Belmonte et al. | Brains, genes, and primates | |
| Gosden et al. | Portrait of an oocyte: our obscure origin | |
| Bianco-Miotto et al. | Recent progress towards understanding the role of DNA methylation in human placental development | |
| Hammer et al. | Production of transgenic rabbits, sheep and pigs by microinjection | |
| CN1250715C (en) | Unactivated oocytes as cytoplast recipients for nuclear transfer | |
| Germond et al. | Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 μm diode laser beam | |
| KR102149010B1 (en) | Production of parthenogenetic stem cells and patient-specific human embryonic stem cells using somatic cell nuclear transfer | |
| Sofikitis et al. | Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development | |
| CN1202084A (en) | Quiescent cell populations for nuclear transfer | |
| Takahashi et al. | Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes | |
| Tsai et al. | Development of gonadal tissue and aromatase function in the protogynous orange-spotted grouper Epinephelus coioides | |
| Brun | Studies on fertilization in Xenopus laevis | |
| KR100868894B1 (en) | Method for establishing gonochoristic shellfish selfing line | |
| Park et al. | Assisted reproductive techniques and genetic manipulation in the common marmoset | |
| Kaufman | The experimental production of mammalian parthenogenetic embryos | |
| Hayashi et al. | Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl | |
| BURGOS et al. | The effects of purified gonadotrophins on the morphology of the testes and thumb pads of the normal and hypophysectomized autumn frog (Rana pipiens) | |
| CN109820819B (en) | Preparation and application method of improved injection mixed solution | |
| Chen et al. | Recovery of functional oocytes from cultured premeiotic germ cells after kidney capsule transplantation | |
| DE60133644T2 (en) | EMBRYONAL STEM CELLS ABOLISHED FROM APES | |
| Colombo et al. | Gonad sex differentiation of Anguilla anguilla by sex steroids | |
| Mitalipov et al. | Nuclear transfer in nonhuman primates | |
| Senbon et al. | Fertilization and development of bovine oocytes grown in female SCID mice | |
| Wakayama et al. | Production of cloned mice from somatic cells, ES cells, and frozen bodies | |
| CN107312749B (en) | Physical method for activating oocyte and evaluation index of fertilization potential of oocyte |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210323 |