CN107779428A - A kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source - Google Patents
A kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source Download PDFInfo
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- CN107779428A CN107779428A CN201711034939.XA CN201711034939A CN107779428A CN 107779428 A CN107779428 A CN 107779428A CN 201711034939 A CN201711034939 A CN 201711034939A CN 107779428 A CN107779428 A CN 107779428A
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- ridge
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- stem cell
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source, belong to biological technical field, comprise the following steps：（1）21 24HH body early embryos of sterile taking-up, are embathed 3 times using PBS；（2）Go down end to end in disecting microscope, separate sex-ridge, obtain early stage sex-ridge tissue, embathed 3 times using PBS；（3）The trypsase for adding preheating is digested, and room temperature digestion 5mins, gently blowing and beating during digestion makes sex-ridge tissue dispersion；（4）Add the culture medium containing calf serum and terminate digestion, 300 leave heart 5mins after 100 mesh screen filtrations, are resuspended after abandoning supernatant using PGCs culture mediums, and differential velocity adherent culture is carried out after resuspension, suct clear after culture 45mins, the higher PGCs of purity is can obtain after continuous differential adhere-wall culture 3 times.Present invention extraction is easy to be quick, it is intended to from early stage sex-ridge（21‑24HH）Middle separation lot of pure and the high PGCs of totipotency, lay the foundation for PGCs application.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of new separation and culture chicken embryo sex-ridge source it is original
Germline stem cell method.
Background technology
Archaeocyte (Primordial germ cells, PGCs) is the beginning of reproduction cell at different levels and ripe gamete
Progenitor cells, reproduction cell are the precursors of sperm and egg cell.During embryo and ontogeny, PGCs is by continuous
Division and differentiation develop into sperm and egg cell.PGCs is a kind of cell with development multipotency, in stem-cell research, is turned
Play an important roll in genetic animal and chimera production and Developmental Biology research.At present both at home and abroad to the side of chicken PGCs separation
Method is largely reported.Mainly there is germinal crescent to separate (5-8HH) at present, embryo's blood separation (12-19HH) and gonad point
From (28HH), separation method can also influence PGCs separating effect.Han Yi ice et al. is by separating hatching to 48-55 hour chicken embryos
(5-8HH) blood, separation obtain PGCs；Li Zandong et al. has used Ficoll density level bands from 12-17 phase chicken embryo blood
Tri- kinds of degree centrifugation, filter membrane and MiniMACS methods have separated PGCs, compare the effect of three kinds of methods；The explorations such as Qin Jie pass through
Ficoll density gradient centrifugations and trypsin digestion separate (28HH) with gonad in embryo's blood separation (12-19HH) respectively
PGCs separating effects.Substantial amounts of research shows that it is more wide that embryo's blood separation (12-19HH) separates (28HH) application with gonad
It is general, and PGCs can be obtained and be used to study in next step, however, it was found that the PGCs negligible amounts of blood separation, and gonad separation
PGCs contains the cell in other a large amount of sources, and purifying is complex.In addition, PGCs vitro culture conditions require complicated cell
Cultivating system, ensure that it possesses the ability of infinite multiplication and undifferentiated state, different separation methods is taken in different experiments room
And system, because PGCs versatility support mechanism is still not clear, and various method complex operations, poor repeatability, limit
PGCs further application is, it is necessary to further optimize and improve.
The content of the invention
The purpose of the present invention is to overcome prior art shortcoming, there is provided a kind of new separation chicken embryo sex-ridge source it is original
Germline stem cell method, extract easy to be quick, it is intended to lot of pure is separated from early stage sex-ridge (21-24HH) and totipotency is high
PGCs, laid the foundation for PGCs application.
The purpose of the present invention is achieved through the following technical solutions, a kind of new separation chicken embryo sex-ridge source it is original
Germline stem cell method, comprises the following steps：
(1) sterile taking-up 21-24HH body early embryos, are embathed 3 times using PBS；
(2) go down end to end in disecting microscope, separate sex-ridge, obtain early stage sex-ridge tissue, embathe 3 using PBS
It is secondary；
(3) trypsase for adding preheating (37 DEG C) is digested, and room temperature digestion 5mins, gently blowing and beating during digestion makes life
Grow ridge tissue dispersion；
(4) add the culture medium containing calf serum and terminate digestion, 300 leave heart 5mins after 100 mesh screen filtrations, abandon
It is resuspended after supernatant using PGCs culture mediums, differential velocity adherent culture is carried out after resuspension, is sucted clearly after cultivating 45mins, it is continuous poor
The higher PGCs of purity is can obtain after fast adhere-wall culture 3 times.
Preferably, the sex-ridge position is before body early embryo hindgut 1/3 back of the body outside tissue position.
Preferably, the mass concentration of the trypsase is 0.25%, and the addition volume of trypsase is 1-2ml.
Preferably, the mass concentration of the culture medium containing calf serum is 5%, the culture medium containing calf serum
Volume is 1-2ml.
Preferably, the preheating temperature of the trypsase is 37 DEG C.
The present invention is advantageous in that：
1. the PGCs in the present invention by blood from just sex-ridge is moved into, than the PGCs after the complete colonization of sex-ridge
With higher proliferation activity and versatility.
2. the PGCs obtained in the present invention, compared with blood sources PGCs, the interference such as haemocyte is less and simple to operate, weight
Renaturation is good, and the requirement to instrument and equipment facility is relatively low, has extensive promotion prospect.
3. the present invention uses co-culture method, without specially building feeder layer, cell factor and its are added in the medium
His nutritional ingredient, it is able to maintain that PGCs undifferentiated state and multiplication capacity.
4. the present invention can with fast and convenient acquisition largely with versatility and the strong PGCs of multiplication capacity, can be used for into
The experimental study of one step, for PGCs build be culture, transgenic chicken prepare and chimera research etc. popularization and application establishes
Good basis.
Brief description of the drawings
Fig. 1 body early embryos (21-24HH) gonad sample position schematic diagram；
Fig. 2A is the PGCs form culture figures just separated；
Fig. 2 B are the PGCs form culture figures of agglomerate growth after long-term cultivation；
Fig. 3 AKP coloration results；
Fig. 4 A are SSEA-1 qualification results figures (details in a play not acted out on stage, but told through dialogues)；
Fig. 4 B are SSEA-1 qualification results figures (light field)；
The RT-PCR qualification figures of Fig. 5 PGCs specific genes.
Embodiment
A kind of primordial germ stem cell methods in new separation chicken embryo sex-ridge source, comprise the following steps：
(1) collection and cleaning of body early embryo (21-24HH)
Embryo egg normal incubation successively pricks bromine phenol and 75% alcohol washes embryo to 3.5 days to 4.5 days (21-24HH) using benzene
Egg, embryo egg is opened by blunt end after cleaning, embryo is gently gripped using tweezers, be placed in the plate for filling PBS (no calcium ions and magnesium ions)
Cleaned, embryo is cleaned three times using PBS respectively, washes away the debris such as yolk.
(2) acquisition of early stage sex-ridge tissue
Go using ophthalmic tweezers end to end, gently to separate sex-ridge under disecting microscope, sex-ridge position is 1/3 before hindgut
Back of the body outside tissue position (Fig. 1).The early stage sex-ridge tissue of acquisition is put into the cillin bottle for filling PBS (no calcium ions and magnesium ions).
(3) digestion of early stage sex-ridge tissue
To the sex-ridge tissue obtained in (2), upper strata PBS is gently sucked, adds the trypsase of 1-2ml preheatings
(0.25%) digested, room temperature digestion 5mins, gently being blown and beaten using pipettor or sterile glass pipette during digestion makes reproduction
Ridge tissue dispersion.
(4) PGCs separation
To obtaining digestive juice in (3), add the culture medium that 1-2ml contains calf serum (5%) and terminate digestion, 100 mesh nets
300 leave heart 5mins after sieved filter, are resuspended after abandoning supernatant using PGCs culture mediums, and differential velocity adherent culture is carried out after resuspension,
Clear liquid is sucted after culture 45mins is placed i.e. in cell culture incubator, continuously takes 3 supernatants to divide other a large amount of cells
From obtaining the higher PGCs of purity.
PGCs medium components are as follows：
(5) PGCs culture and identification
Purifying PGCs cells can be obtained by cultivating 10d or so to the PGCs obtained in (4), and now cell shape is more in ellipse
The irregular projections such as shape, small hill shape, nest like, the characteristic (Fig. 2A, 2B) of agglomerate growth can be presented.SSEA-1 mirror are carried out to cell
Fixed (Fig. 3), AKP dyeing (Fig. 4 A, 4B) and RT-PCR identifications (Fig. 5), the gene of identification include NANOG, OCT4, CVH, DAZL with
KIT。
RT-PCR primer is as follows：
Claims (5)
- A kind of 1. primordial germ stem cell methods for separating chicken embryo sex-ridge source, it is characterised in that：Comprise the following steps：（1）Sterile taking-up 21-24HH body early embryos, are embathed 3 times using PBS；（2）Go down end to end in disecting microscope, separate sex-ridge, obtain early stage sex-ridge tissue, embathed 3 times using PBS；（3）The trypsase for adding preheating is digested, and room temperature digestion 5mins, gently blowing and beating during digestion makes sex-ridge tissue point Dissipate；（4）Add the culture medium containing calf serum and terminate digestion, 300 leave heart 5mins after 100 mesh screen filtrations, abandon supernatant It is resuspended afterwards using PGCs culture mediums, differential velocity adherent culture is carried out after resuspension, clear, continuous differential patch is sucted after cultivating 45mins The higher PGCs of purity is can obtain after wall culture 3 times.
- 2. a kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source according to claim 1, its feature exist In：The sex-ridge position is before body early embryo hindgut 1/3 back of the body outside tissue position.
- 3. a kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source according to claim 1 or 2, its feature It is：The mass concentration of the trypsase is 0.25%, and the addition volume of trypsase is 1-2ml.
- 4. a kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source according to claim 3, its feature exist In：The mass concentration of the culture medium containing calf serum is 5%, and the volume of the culture medium containing calf serum is 1-2ml.
- 5. a kind of primordial germ stem cell methods for separating chicken embryo sex-ridge source according to claim 4, its feature exist In：The preheating temperature of the trypsase is 37 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925981A (en) * | 2020-08-26 | 2020-11-13 | 扬州大学 | Method for separating chicken male and female primordial germ cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005026326A2 (en) * | 2003-09-10 | 2005-03-24 | Tranxenogen, Inc. | Generation of chicken cell lines from embryonic stem cells and germ cells |
| CN105779377A (en) * | 2016-04-05 | 2016-07-20 | 佛山科学技术学院 | Method for separating original germ stem cells with chicken embryo blood source |
| CN105838791A (en) * | 2016-04-21 | 2016-08-10 | 扬州大学 | Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells |
| US20170086431A1 (en) * | 2005-02-01 | 2017-03-30 | Alexion Pharma Llc | Germline transmission of chicken primordial germ cells (pgcs) |
-
2017
- 2017-10-30 CN CN201711034939.XA patent/CN107779428A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005026326A2 (en) * | 2003-09-10 | 2005-03-24 | Tranxenogen, Inc. | Generation of chicken cell lines from embryonic stem cells and germ cells |
| US20170086431A1 (en) * | 2005-02-01 | 2017-03-30 | Alexion Pharma Llc | Germline transmission of chicken primordial germ cells (pgcs) |
| CN105779377A (en) * | 2016-04-05 | 2016-07-20 | 佛山科学技术学院 | Method for separating original germ stem cells with chicken embryo blood source |
| CN105838791A (en) * | 2016-04-21 | 2016-08-10 | 扬州大学 | Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells |
Non-Patent Citations (2)
| Title |
|---|
| XINYAN TANG等: "Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells", 《ANIMAL REPRODUCTION SCIENCE》 * |
| 李碧春等: "鸡胚PGCs迁移与性腺发育关系的研究", 《扬州大学学报（农业与生命科学版）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925981A (en) * | 2020-08-26 | 2020-11-13 | 扬州大学 | Method for separating chicken male and female primordial germ cells |
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