CN113249303B - Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells - Google Patents
Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells Download PDFInfo
- Publication number
- CN113249303B CN113249303B CN202110497403.1A CN202110497403A CN113249303B CN 113249303 B CN113249303 B CN 113249303B CN 202110497403 A CN202110497403 A CN 202110497403A CN 113249303 B CN113249303 B CN 113249303B
- Authority
- CN
- China
- Prior art keywords
- primordial germ
- vegf
- germ cells
- chicken
- proliferation
- 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.)
- Active
Links
Images
Classifications
-
- 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
- C12N5/0608—Germ cells
- C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
Abstract
The invention belongs to the technical field of poultry breeding and reproductive management, and particularly relates to application of a vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells. The effect of Vascular Endothelial Growth Factor (VEGF) on the proliferation and migration capacity of chicken primordial germ cells was compared. VEGF with proper concentration is added on the basis of the existing chicken primordial germ cell culture solution, the proliferation of chicken primordial germ cells can be promoted, the proliferation promoting effects on the chicken primordial germ cells are different due to different VEGF adding concentrations, and the application effect is optimal when the intravascular VEGF concentration is 25ng/mL and the chicken primordial germ cells are treated for 3 days. The invention can effectively promote the proliferation of the chicken primordial germ cells in vitro, obviously improve the capacity of migrating and fixedly planting the chicken primordial germ cells by VEGF with proper concentration in the basic culture solution, and maintain the biological characteristics unchanged.
Description
Technical Field
The invention belongs to the technical field of poultry breeding and reproduction management. In particular to the application field of Vascular Endothelial Growth Factor (VEGF) in promoting the proliferation of chicken primordial germ cells and improving the gonad migration and colonization capacity.
Background
Primordial Germ Cells (PGCs) are progenitor cells that give rise to sperm and oocytes and can transmit genetic information from parent to offspring. Currently, the most efficient and feasible way to produce transgenic chickens is to make in vitro modification editing on chicken primordial germ cells and then reinject embryonic blood vessels to allow them to migrate into gonads to develop into gametes (Arjun Challagula et al 2021.Marker counter-selection via CRISPR/Cas9 co-targeting for efficacy generation of gene-edited average cell lines and germ cells. animal biotechnol. Mar 2: 1-11.). In addition, due to the particularity of the hatching eggs, a large amount of egg yolks are stored, the egg yolks cannot be stored in vitro for a long time, and the chicken primordial germ cells have wide application prospects in the aspect of conservation of rare variety resources. In chickens, PGCs first appear in the central region of the X-stage embryo, then enter the vascular circulation system, and finally migrate into the early gonads for colonization. However, the small number of PGCs isolated from early embryos, which allows the proliferation of a limited number of PGCs in vitro culture without loss of the potential of germ stem cells and which can improve the migration efficiency, is a problem that needs to be solved urgently for the production of gene-edited chickens and for future work of breed conservation and transplantation. Therefore, it is essential to establish an in vitro culture system which promotes the efficient proliferation of chicken primordial germ cells and can improve the migration efficiency.
Several methods for the isolation and in vitro culture of chicken primordial germ cells have been successful. Robert J et al, which extracted a small amount of blood from 14-17 stage chick embryos, cultured on a medium based on KO-DMEM with rat hepatocyte cells as a feeder layer, successfully isolated and cultured primordial germ cells capable of Germline transmission for a long period of time (Van de Lavoir et al 2006. Germinline transmission of genetic modified primordial germ cells. Nature, 441(7094), 766 769.). In a similar manner, Han et al also successfully isolated and cultured chicken primordial germ cells in vitro in another medium based on KO-DMEM, and maintained the cellular characteristics of primordial germ cells and had gonad-migratory capacity (Park and Han 2012.piggyBac transfer in primordial germ cells is an infection tool for transgenesis in chicken. Proc Natl Acad Sci USA 109(24), 9337-9341.). However, these methods are still unstable and inefficient.
Vascular Endothelial Growth Factor (VEGF), a highly specific, endothelin, binds to specific high affinity transmembrane receptors expressed predominantly on Endothelial cells to promote increased Vascular permeability, extracellular matrix degeneration, Vascular Endothelial cell migration, proliferation, and angiogenesis, and can also exert its biological functions by binding to tyrosine kinase receptors 1(Flt-1) and 2(Flk/KDR) also located on the surface of stem cells. VEGF also stimulates the growth, survival and proliferation of vascular endothelial cells. VEGF is a family of structurally related proteins including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor. VEGF-A, abbreviated VEGF, is the most important factor in this family that induces the migration and division of endothelial cells and activates angiogenesis. The research shows that the Vascular Endothelial Growth Factor (VEGF) and the receptor thereof (VEGFR) are important regulating factors for the growth, development and differentiation of heart and blood vessels, have the effects of neurotrophic and neuroprotection to neurons and glial cells in the normal growth and development process of organisms, and can promote the proliferation and survival of neural stem cells. Whether the primordial germ cells of the chicken enter blood at the 10 th stage of embryonic development and whether vascular endothelial cell growth factors have influence on the proliferation and migration of the primordial germ cells of the chicken, and how different VEGFA subtypes participate in the biological process of stem cells in the embryonic development process still needs to be determined.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of Vascular Endothelial Growth Factor (VEGF) in promoting proliferation of chicken primordial germ cells and improving the gonad migration and colonization capacity.
The technical scheme of the invention is as follows:
the application of vascular endothelial cell growth factor (VEGF) in promoting the proliferation and migration capacity of chicken primordial germ cells for non-diagnosis purpose is characterized in that the vascular endothelial cell growth factor is used by the following culture medium combination, and the specific steps are as follows: (1) preparation of VEGF basic culture solution
KO-DMEM: 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate, 100ug/mL heparin sodium, 1X antibody-agonist, 1X GlutaMax, 1X GS nucleotide supplement, 0.1mM beta-mercaptoethanol, 10ng/mL hFGFb, 1X NEAA, 1X B-27supplement and 25ng/mL Human activator A, and (2) preparation of VEGF culture solution
KO-DMEM: 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate, 100ug/mL heparin sodium, 1X antibody-agonist, 1X GlutaMax, 1X GS nucleotide supplement, 0.1mM beta-mercaptoethanol, 10ng/mL hFGFb, 1X NEAA, 1X B-27supplement, 25ng/mL Human Activin A, the use concentration of vascular endothelial cell growth factor is 25 ng/mL-50 ng/mL.
Preferably, the optimal concentration of vascular endothelial growth factor in VEGF culture is 25 ng/mL.
Chick primordial germ cells, which have developmental potential as precursor cells for sperm and eggs, are isolated from chick embryo blood where the amount of Vascular Endothelial Growth Factor (VEGF) VEGF is indeterminate. To this end, the inventors have intensively studied the effect of VEGF on the proliferation and migration of chicken primordial germ cells. The result shows that VEGF is added on the basis of the existing culture solution for culturing the chicken primordial germ cells, so that the proliferation of the chicken primordial germ cells can be promoted, and the migration efficiency of the chicken primordial germ cells can be improved. According to the difference of the added VEGF concentration, the influence degree of the VEGF on the proliferation of the chicken primordial germ cells is different, when the VEGF concentration is 25ng/mL, the effect is most obvious, the proliferation and migration of the chicken primordial germ cells in vitro can be effectively promoted, and the biological properties of the chicken primordial germ cells can be well maintained in a culture solution containing the VEGF at the concentration.
Primordial germ cells are a genetic resource with great application prospect, however, when the primordial germ cells of chicken are cultured in vitro by using a traditional method, the primordial germ cells proliferate slowly and gradually lose germ line capability. Therefore, the invention adds the vascular endothelial growth factor into the traditional culture system, optimizes and develops the culture system for stably proliferating the chicken primordial germ cells in vitro, and improves the proliferation capacity and the migration capacity of the chicken primordial germ cells.
Drawings
FIG. 1: effect of different concentrations of VEGF on PGC proliferation. Description of reference numerals: VEGF was graded at final concentrations of 0, 25 and 50ng/mL, and OD measured at 450nm of the supernatant after PGC treatment at 1d, 3d and 5d to obtain a line graph.
FIG. 2: cultured cells were identified by immunofluorescence staining. Description of reference numerals: panel A of FIG. 2, a bright field image of SSEA-1 stained cells of chicken primordial germ cells; panel B in fig. 2, SSEA1 immunofluorescence positive cells; panel C in fig. 2, immunofluorescence staining of nuclei with DAPI; FIG. 2, panel D, graph for targeting of DAPI and SSEA-1 staining. FIG. 2 is a photograph showing the staining of negative control chicken fibroblast line DF-1 in the fourth panel of the upper half of FIG. 2, wherein: FIG. 2 is E-plot of the light field of the negative control cells; panel F in figure 2 is a negative control cell immunofluorescent stain; FIG. 2 is a G-plot of immunofluorescence staining of cell nuclei with DAPI; FIG. 2 is a graph H showing the staining of large with DAPI and SSEA-1. FIG. 2 is a graph I showing a brightfield of cells stained with DDX4, which is a chicken primordial germ cell; panel J in figure 2 is DDX4 immunofluorescence positive cells; FIG. 2 is K-plot of immunofluorescence staining of cell nuclei with DAPI; the L plot in FIG. 2 is the Merge plot of DAPI and DDX4 staining. The four panels in the second row of the lower half of FIG. 2, namely, M, N, O and P, are staining pictures of the negative control chicken fibroblast line DF-1, respectively. Wherein the M plot in FIG. 2 is a light field plot of the negative control cells; FIG. 2 is a graph N showing immunofluorescence staining of negative control cells; FIG. 2 is O-plot of immunofluorescent staining of cell nuclei with DAPI; the P plot in fig. 2 is the Merge plot of DAPI and DDX4 staining.
FIG. 3: cell map of chicken primordial germ cells after PKH26 fluorescence labeling. Description of reference numerals: FIG. 3A is a brightfield image of chicken primordial germ cells after fluorescent labeling with PKH 26; the B picture in FIG. 3 is a red fluorescence picture of chicken primordial germ cells after fluorescent labeling with PKH 26.
FIG. 4: the identification picture of the gonad migration ability of the cultured chicken primordial germ cells. Description of reference numerals: FIG. 4A is a graph showing the migration of red fluorescent PGCs from a control group of 6 day old recipient chick embryos; the B graph in FIG. 4 is a graph of migration of red fluorescent PGCs in the VEGF-treated group of gonads of 6-day-old recipient chick embryos, and the C graph in FIG. 4 is a histogram of statistics of the number of migration of red fluorescent PGCs in the control group and the VEGF-treated group.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples.
The methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of culture solution
(1) VEGF basal medium:
KO-DMEM, 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate (from Beijing Soulebao technologies, Inc.), 100ug/ml heparin sodium (from Beijing Soulebao technologies, Inc.), 1X anti-biological-antifungal (antibacterial-antifungal from Seimer Feishehell technologies, Inc.), 1X GlutaMax (glutamine additive from Seimer Feishao technologies, Inc.), 1X GS nucleotide supplement from Mercury Miche, Inc.), 0.1mM beta-mercaptoethanol, 10ng/ml hFGFb (human basic fibroblast growth factor from Beijing Soulebao technologies, Inc.), 1X NEAA (non-essential amino acid solution from Seimer Feishao technologies, Inc.), 1-27 supplement from 1X B-27 (B-27 supplement from Beijing Soulebao technologies, Inc.), 25ng/mL Human Activin A (Human Activin A, available from Papultak Biotech, Inc.).
(2) VEGF culture solution:
KO-DMEM, 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate (from Beijing Soulebao technologies, Inc.), 100ug/ml heparin sodium (from Beijing Soulebao technologies, Inc.), 1X anti-biological-antimycotic (antibacterial-antifungal from Seimer Feishehell technologies, Inc.), 1X GlutaMax (glutamine additive from Seimer Feishao technologies, Inc.), 1X GS nucleotide supplement (nucleoside supplement from Mercury Miche, Inc.), 0.1mM beta-mercaptoethanol, 10ng/ml hFGFb (human basic fibroblast growth factor from Beijing Soulebao technologies, Inc.), 1X NEAA (non-essential amino acids from Seimer Feishao technologies, Inc.), 1X 1X B-27 Susus (B-27 supplement from B-X B, from Beijing Soulebao technologies, Inc.), 25ng/mL Human Activin A (Human Activin A, available from Peptake Biotech, Inc.), VEGF (vascular endothelial growth factor, available from Beijing Soilebao Tech., Ltd.) 25ng/mL-50 ng/mL.
Example 2 Chicken primordial germ cell culture and VEGF treatment
The breeding eggs of the red Yao chicken (produced in Jiangsu, the practice of the present invention is not limited to this variety) used for the test were incubated at 37.8 ℃ and a relative humidity of 60% (using a commercial chick embryo incubator) until 14-17HH stage (incubation period of 60 hours), the eggshells were sterilized with alcohol, the eggs were opened and placed in a petri dish, 5-10. mu.L of blood from the yolk vein, heart, etc. was collected by siphoning with a glass pipette, and the blood was added to a 1.5mL centrifuge tube containing PBS (phosphate buffer, purchased from Saimer Feishell science Co., Ltd.). Transferring the isolated chicken primordial germ cells into 24-well cell culture plate, adding freshly prepared VEGF basic culture solution, standing at 39 deg.C and containing 5% CO 2 The culture box is used for culturing for 3 days, half of liquid is changed every other day, and subculture can be carried out after 5 days.
(1) Inoculating chicken primordial germ cells in logarithmic growth phase into 96-well plate, and adding VEGF-containing solution into the well to final concentration gradientThe culture broth (3 replicates per concentration gradient) was prepared at 0, 25, 50ng/mL, while an equal amount of basal medium without VEGF was added to the control group. The chicken primordial germ cells are treated at 39 ℃ with 5% CO 2 Cultured in an incubator.
Example 3 cell proliferation assay
(1) The chicken primordial germ cells in the 96-well plate were cultured for 1d, 3d, and 5d after the VEGF addition treatment, and then incubated for 2 hours by adding 10. mu.L of CCK-8 mixture (purchased from Beijing Boaoson Biotechnology Co., Ltd.) respectively.
(2) The optical density of the supernatant was measured at 450 nm. The test results are shown in FIG. 1. The invention finds that the proliferation rate and the survival rate of chicken primordial germ cells can be improved by using 25ng/mL and 50ng/mL VEGF treatments when culturing for 1-3d, wherein the proliferation promoting effect of 25ng/mL VEGF is very obvious, and the proliferation rate and the survival rate of chicken primordial germ cells can be inhibited to a certain extent by using 50ng/mL VEGF for 5d, so that the 25ng/mL VEGF is determined as the optimal treatment concentration of the culture system in the embodiment, and the 3d culture is determined as the optimal treatment time of the culture system.
Example 4 immunofluorescent staining identification of primordial germ cells
(1) The cultured primordial germ cells are fixed by paraformaldehyde solution (4% PFA), washed by Phosphate Buffer Solution (PBS) for 3 times, treated by Triton X-100 for 10min, treated by goat serum (purchased from Beijing Solebao science and technology Co., Ltd.) blocking solution for 30min, incubated by SSEA-1 (purchased from DSHB antibody Co., MC-480, in a volume ratio of 1:200) antibody diluent at 4 ℃ overnight, washed by PBS for 3 times, added with goat anti-mouse secondary antibody (purchased from Saimer Feishell science and technology Co., Ltd., A-21042, in a volume ratio of 1:200) at room temperature for 1h, added with DAPI, incubated in dark for 5min, and observed under a fluorescence microscope. It can be seen that the cell membrane of the isolated chicken primordial germ cell expresses SSEA-1 primordial germ cell specific marker, i.e. green fluorescence is present, and the cell nucleus is DAPI stained (see FIG. 2).
(2) The cultured chicken primordial germ cells are fixed by paraformaldehyde solution (4% PFA), rinsed for 3 times by PBS buffer, treated for 10min by Triton X-100, treated for 30min by goat serum blocking solution, incubated at 4 ℃ overnight by DDX4 (purchased from Ebos Shanghai Co., Ltd., ab13840, volume ratio used is 1:200) antibody diluent, rinsed for 3 times by PBS, incubated for 1h at room temperature by adding goat anti-rabbit secondary antibody (purchased from Wuhan Saville Biotech Co., Ltd., GB25303, volume ratio used is 1:200), incubated for 5min by adding DAPI, and observed under a fluorescence microscope. It can be seen that the cell membrane of the isolated chicken primordial germ cell expresses a specific marker DDX4 primordial germ cell, and the cell nucleus shows green fluorescence and DAPI staining (see figure 2).
Example 5 identification of the gonadal migration ability of chicken primordial germ cells
The test group of chicken primordial germ cells were treated with medium containing 25ng/mL VEGF for 3d, while the control group of chicken primordial germ cells were treated with medium containing no VEGF for 3 d. The chicken primordial germ cells are subjected to red fluorescence labeling by using a fluorescent dye PKH26 before microinjection, the labeling efficiency is close to 100 percent (shown in figure 3) through fluorescence microscope observation, and then the cells are collected for chick embryo injection.
Chick embryo injection is carried out according to the following steps: take 1X 10 6 Primordial germ cells were resuspended in 50. mu.L of culture medium, 2. mu.L of cell suspension was aspirated with a microinjection glass needle and injected into the dorsal aorta where 60h chick embryos were incubated. Chick embryos were dissected the fourth day after injection and the number of fluorescent cells in the gonads was observed. FIG. 4 shows the result of the identification of gonad migration ability of chicken primordial germ cells, and it can be seen that the primordial germ cells cultured and fluorescently labeled with red have the ability to successfully migrate into gonads, and the number of cells migrated is greater for PGCs treated with 25ng/mL VEGF than for PGCs of the control group, so that PGCs treated with 25ng/mL VEGF have stronger migration ability.
In conclusion, the VEGF concentration is 25ng/mL, the effect is most obvious, the treatment time is 3d, and the optimal technical scheme of the culture system is provided, so that the in-vitro proliferation efficiency of the chicken primordial germ cells can be effectively promoted, the biological characteristics of the chicken primordial germ cells can be well maintained in a culture solution containing 25ng/mL of VEGF, and the capability of the chicken primordial germ cells in migrating and fixedly planting gonads can be obviously improved.
Claims (2)
1. The application of Vascular Endothelial Growth Factor (VEGF) in promoting the proliferation and migration capacity of chicken primordial germ cells for non-diagnostic purposes is characterized in that the application step is that the vascular endothelial growth factor is combined by the following culture media: (1) preparing VEGF basic culture solution:
KO-DMEM: 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate, 100ug/mL heparin sodium, 1X antibody-agonist, 1X GlutaMax, 1X GS nucleotide supplement, 0.1mM beta mercaptoethanol, 10ng/mL hFGFb, 1X NEAA, 1X B-27supplement, and 25ng/mL Human activator A, and
(2) preparing a VEGF culture solution:
KO-DMEM: 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate, 100ug/mL heparin sodium, 1X antibody-agonist, 1X GlutaMax, 1X GS nucleotide supplement, 0.1mM beta-mercaptoethanol, 10ng/mL hFGFb, 1X NEAA, 1X B-27supplement, 25ng/mL Human Activin A, vascular endothelial cell growth factor (VEGF) concentration of 25ng/mL-50 ng/mL.
2. The use of a VEGF of claim 1 in the preparation of a medicament for promoting the proliferation and migration of chicken primordial germ cells, wherein the VEGF is used at a concentration of 25 ng/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110497403.1A CN113249303B (en) | 2021-05-07 | 2021-05-07 | Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110497403.1A CN113249303B (en) | 2021-05-07 | 2021-05-07 | Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113249303A CN113249303A (en) | 2021-08-13 |
| CN113249303B true CN113249303B (en) | 2022-07-29 |
Family
ID=77223883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110497403.1A Active CN113249303B (en) | 2021-05-07 | 2021-05-07 | Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113249303B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586944B (en) * | 2024-01-19 | 2024-04-30 | 成都思澍生物科技有限公司 | Method for in-vitro serum-free amplification culture of primordial germ cells of chickens |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107189980A (en) * | 2005-06-22 | 2017-09-22 | 阿斯特利亚斯生物治疗股份公司 | The suspension culture of human embryo stem cell |
| CN108384749A (en) * | 2017-12-07 | 2018-08-10 | 广西大学 | Chicken sexual gland archaeocyte quick separating and build the method for being |
| CN111484971A (en) * | 2019-05-15 | 2020-08-04 | 林柳吟 | Preparation method, kit and application of blood-derived female autologous reproductive stem cells |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0220145D0 (en) * | 2002-08-30 | 2002-10-09 | Thromb X Nv | Novel compositions for the in vitro derivation and culture of embryonic stem (ES) cell lines with germline transmission capability |
| WO2005038014A1 (en) * | 2003-10-17 | 2005-04-28 | Innovative Dairy Products Pty Ltd As Trustee For The Participants Of The Cooperative Research Centre For Innovative Dairy Products | Isolation of stem cell-like cells and use thereof |
| CA2570086A1 (en) * | 2004-06-10 | 2005-12-29 | Merial Limited | Medium and methods for culturing of avian primordial germ cells |
| US8652845B2 (en) * | 2010-03-18 | 2014-02-18 | Kyoto University | Method for producing mesodermal cells by culturing under adherent conditions and without co-culture with cells from a different species in a serum-free medium |
-
2021
- 2021-05-07 CN CN202110497403.1A patent/CN113249303B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107189980A (en) * | 2005-06-22 | 2017-09-22 | 阿斯特利亚斯生物治疗股份公司 | The suspension culture of human embryo stem cell |
| CN108384749A (en) * | 2017-12-07 | 2018-08-10 | 广西大学 | Chicken sexual gland archaeocyte quick separating and build the method for being |
| CN111484971A (en) * | 2019-05-15 | 2020-08-04 | 林柳吟 | Preparation method, kit and application of blood-derived female autologous reproductive stem cells |
Non-Patent Citations (7)
| Title |
|---|
| Basic Fibroblast Growth Factor Activates MEK/ERK Cell Signaling Pathway and Stimulates the Proliferation of Chicken Primordial Germ Cells;Choi, JW等;《PLOS ONE》;20100923;第5卷(第9期);第1-11页 * |
| Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2;Daichi MIYAHARA等;《Journal of Reproduction and Development》;20151228;第62卷(第2期);第143-149页 * |
| Vascular endothelial growth factor A: just one of multiple mechanisms for sex-specific vascular development within the testis?;Sargent, KM等;《Journal of Endocrinol》;20151118;第227卷(第2期);第R31-R50页 * |
| Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis;Caires, K. C.等;《Reproduction》;20090101;第138卷(第4期);第667-677页 * |
| VEGF在人胎卵巢发育过程中的表达研究;艾庆燕等;《中国组织化学与细胞化学杂志》;20130830;第22卷(第4期);第301-305页 * |
| 鸡生殖干细胞研究现状与展望;李碧春等;《中国家禽》;20110605;第33卷(第11期);第1-6页 * |
| 鸡胚胎原始生殖细胞的培养和传代;秦洁等;《动物学报》;20050815;第51卷(第4期);第723-731页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113249303A (en) | 2021-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Staub | A century of research on mammalian male germ cell meiotic differentiation in vitro | |
| RU2473688C2 (en) | Method for producing transgenic birds using embryo stem cells | |
| CN104350145B (en) | The method of stem spermatogonium for no feeder cells culture ox and pig | |
| CN108384749B (en) | Method for quickly separating and establishing chicken gonad primordial germ cells | |
| Raucci et al. | In vitro generation and characterization of chicken long-term germ cells from different embryonic origins | |
| CN113249303B (en) | Application of vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells | |
| KR100569168B1 (en) | Method for Culturing Avian Spermatogonial Stem Cells and Avian Spermatogonial Stem Cells Prepared thereby | |
| JP2003532364A (en) | Avian primordial germ cell (PGC) cell line and long-term culture method | |
| Akhlaghpour et al. | Chicken interspecies chimerism unveils human pluripotency | |
| Xie et al. | Derivation of chicken primordial germ cells using an indirect Co-culture system | |
| Mozdziak et al. | Production of chick germline chimeras from fluorescence-activated cell-sorted gonocytes | |
| Meng et al. | Identification of oogonial stem cells in chicken ovary | |
| KR100502889B1 (en) | Method for Enhancing Germline Transmission Efficiency of Avian Primordial Germ Cells | |
| Lauschke et al. | A comparative assessment of marker expression between cardiomyocyte differentiation of human induced pluripotent stem cells and the developing pig heart | |
| CN109722411B (en) | Application method of micromolecules for promoting self-renewal state of embryonic stem cells | |
| CN117007813A (en) | FoXO3 gene and application of targeted inhibitor thereof in proliferation and migration colonization of chicken primordial germ cells | |
| US20030115622A1 (en) | Production of avian embryonic germ (eg) cell lines by prolonged culturing of pgc's, use thereof for cloning and chimerization | |
| JP4267689B2 (en) | Method for culturing avian cell and cell line obtained by the culturing method | |
| US20020162134A1 (en) | Primordial germ cell-based germ line production of birds | |
| CA2438612A1 (en) | Primordial germ cell-based germ line production of birds | |
| CN110283779A (en) | A kind of isolated culture method and culture medium of chicken embryonic stem cells | |
| CN117586944B (en) | Method for in-vitro serum-free amplification culture of primordial germ cells of chickens | |
| RU2818641C1 (en) | Method of introducing primordial germ cells of birds into an embryo "in ovo" | |
| KR100267633B1 (en) | Method for production of germline chimeric birds by transfer of cultured primordial germ cell | |
| Sritabtim et al. | First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica) |
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 |