CN101423818A - Method for culturing chicken embryonic germ cells and special nutrient fluid thereof - Google Patents
Method for culturing chicken embryonic germ cells and special nutrient fluid thereof Download PDFInfo
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- CN101423818A CN101423818A CNA2008102400944A CN200810240094A CN101423818A CN 101423818 A CN101423818 A CN 101423818A CN A2008102400944 A CNA2008102400944 A CN A2008102400944A CN 200810240094 A CN200810240094 A CN 200810240094A CN 101423818 A CN101423818 A CN 101423818A
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Abstract
The invention discloses a method for culturing chicken embryonic germ cells and a special culture solution thereof. The culture solution is a high sugar DMEM solution containing the following solutes: 3.5 to 4.5 g/L of glucose, 1.5 to 2.5 mM of L-glutamine, (4.5 to 5.5) x 10<5> M of beta-mercaptoethanol, 1 to 1.5 mM of hydroxyethyl piperazine ethyl sulfuric acid, 100 to 120 IU/ml of leukemia inhibitory factor, 4 to 6 ng/ml of stem cell growth factor, 10 to 12 ng/ml of basic fibroblast growth factor, 10 to 12 ng/ml of insulin-like growth factor-1, 10 to 15 percent of fetal bovine serum, 1 to 3 percent of chicken serum, 1 to 1.2 percent of chick embryo extract, and antibiotic. The method for culturing the chicken embryonic germ cells is to culture the chicken embryonic germ cells on a chicken embryonic fibroblast culture layer. Through the reformation and the modification of EG cells, new varieties of chicken can be effectively cultured.
Description
Technical field
The present invention relates to a kind of method and special culture solution thereof of cultivating the embryonic genital cell of chicken.
Background technology
Embryonic stem cell with multidirectional differentiation potential has two kinds of sources: the one, come from embryonic stem cell (the embryonic stem cell of body early embryo blastaea stage inner cell mass, be called for short the ES cell), another kind is to come from embryonic germ gland primordial germ cells (primordial germ cells, PGCs) embryonic genital cell (embryonicgerm cell is called for short the EG cell).From Evans and Kaufman (1981) from the mouse body early embryo of delayed nidation, separate obtain the ES cell after, body early embryo becomes unique source of separating clone ES cell always.Up to 1992, Matsui etc. were material with attached archeocyte of planting the back mouse fetal, cultivated and obtained the EG cell, and confirmed more all sidedly that first PGCs can be used as ES cellular segregation clone's starting material equally.At present, from PGCs, cultivate the EG cell, on animals such as mouse, rat, pig, people, ox and chicken, obtained important progress.More and more discover, the EG cell in PGCs source is the same with the ES cell, can be used to study the differentiation of early embryo development and cell lineage, and can carry out various genetic manipulations to it, and because the two is very close on form and biological characteristics, so generally again they are referred to as the ES cell.
Generation cycle of chicken is short, and the production performance height is very suitable for the application of transgenic technology.Applying transgene technique is produced pharmaceutical protein as bio-reactor except that carrying out chicken breed improvement, breeding for disease resistance and fundamental research, chicken also has the incomparable advantage of other animal.Yet the fetal development of chicken has been compared a lot of differences with the fetal development of mammal, the ovum of chicken is positioned at the yolk surface, after fertilization is covered by the embryo by male pronucleus, egg is during by output, the embryo has developed into approximately by 60, the blastoderm stage that 000 cell is formed, this makes in Mammals the transgenic technology of widespread use, for example the zygote microinjection is not adapted at using in the bird.Based on the singularity that Embryo Gallus domesticus is grown, present ES cellular pathways is considered to transgenic chicken and makes one of optimal technological line.
The outstanding advantage of ES cell method is to carry out specific genetic modification to the ES cell, the operation of the somatocyte of any formation as intragentic pointed decorations of pair cell such as the retroviral infection of foreign DNA, electricimpulses all applicable to the ES cell.The production performance and the disease-resistant performance that improve bird with the cell-mediated transgenic technology of ES will bring huge economic benefit and social benefit for aviculture.
Also there are all difficulties in the cultivation of EG cell and the detection of karyotype, and a lot of problems wherein await further solution.
Summary of the invention
The purpose of this invention is to provide a kind of method and special culture solution thereof of cultivating the embryonic genital cell of chicken.
The embryonic genital cell nutrient solution of chicken provided by the invention is high sugared DMEM, contains following solute: 3.5-4.5g/L glucose, 1.5-2.5mM L-glutaminate, (4.5-5.5) * 10
-5M beta-mercaptoethanol, 1-1.5mM hydroxyethyl croak piperazine second thiosulfonic acid, 100-120IU/ml (100-120units/ml) leukaemia inhibitory factor, 4-6ng/ml stem cell factor, 10-12ng/ml Prostatropin, 10-12ng/ml insulin-like growth factor-1,10-15% foetal calf serum (volume ratio), 1-3% chicken serum (volume ratio), 1-1.2% chick embryo extract (volume ratio), antibiotic.
Described chick embryo extract can adopt commercially available chick embryo extract also can prepare with the following method:
1) ovum gallinaceum of normal fertilization is hatched in 38 ℃ of incubators or thermostat container.Put a water reservoir in the incubator and keep with the humid air in the case.Stir ovum gallinaceum 1 or 2 times every day.
2) ovum gallinaceum that will hatch 9～12 days was dipped in 95% alcohol 10～15 minutes, take out in the rearmounted small beaker, the air chamber end up, with the eggshell of the tincture of iodine and cotton ball soaked in alcohol sterilization air chamber end.
3) break the eggshell of air chamber end into pieces, peel off eggshell, carefully peel off air bag and chorioallantoic membrane, clamp chicken embryo neck, take out the chicken embryo gently, place plate with the elbow tweezers with tweezers.
4) remove chicken embryo eyes, clot and yolk, with the flushing of Hanks liquid for several times.
5) whole chicken embryo is thoroughly shredded, place tissue homogenizer to grind, or insert in the syringe, pressurization is clamp-oned tissue juice in the centrifuge tube.
6) add the Hanks liquid of organizing equivalent with the chicken embryo, blow and beat mixing gently with suction pipe, sealing was placed in 37 ℃ of thermostat containers 30 minutes, treated the nutritive ingredient leaching in the chicken embryo tissue.
7) the centrifugal 30min of 3000r/min with the packing of sterilization vial, seals.In-20 ℃ of refrigerators, preserve.
Described antibiotic specifically can be penicillin and Streptomycin sulphate; Contain 100-150IU/ml penicillin, 100-120 μ g/ml Streptomycin sulphate in the described nutrient solution.
The definition of 1IU penicillin: 1IU=0.60 μ g Crystalline Penicillin G Sodium salt.
The embryonic genital cell nutrient solution of described chicken specifically contains following solute: 4.5g/L glucose, 2mM L-glutaminate, 5.5 * 10
-5M beta-mercaptoethanol, 1mM hydroxyethyl croak piperazine second thiosulfonic acid, 100IU/ml penicillin, 100ug/ml Streptomycin sulphate, 100IU/ml leukaemia inhibitory factor, 5ng/ml stem cell factor, 10ng/ml Prostatropin, 10ng/ml insulin-like growth factor-1,10% foetal calf serum (volume ratio), 2% chicken serum (volume ratio), 1% (volume ratio) chick embryo extract.
The present invention also provides a kind of method of cultivating the embryonic genital cell of chicken, is that embryonic genital cell with chicken places on the chick embryo fibroblast culture layer and cultivates.
The embryonic genital cell of described chicken is that the embryonic germ gland primordial germ cells cell of the chicken that will be separated to and chicken embryo sexual gland add to cultivate in the described cell culture fluid and obtains.
Described chicken embryo PGCs cell is the 5.5-8.5 embryo cell in age.
The invention provides the method for the embryonic genital cell (EG) of a kind of effective cultivation chicken.PGCs is the precursor cell of gamete, participates in the formation of sexual gland, thereby the embryonic genital cell (EG cell) that utilizes PGCs to cultivate is produced transgenic chicken and had very big advantage.Foreign gene as required positioning integration to the genomic specific position of cell chromosome, by to the transformation of EG cell with modify the new variety that can efficiently cultivate chicken.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
The test materials of using in following examples is as follows:
(1) experimental animal
Chicken is deceived in Shouguang: Liaocheng City is praised bright kind of chicken house (four), and the hair color genotype is CCOOEE, and E is a pigment diffusion gene, and black is to dominance Bai Yuwei recessiveness, and C is a chromogene, and 0 is oxidase gene.
The blue white race egg in sea: available from Eastern Mountain, Tai, Shandong Province kind chicken house (200 pieces), the hair color genotype of extra large blue Cold boiled chicken is IICCOO, and I is the pigment suppressor gene, can cover black and light yellow.
(2) test materials
High sugared DMEM (high glucose): SigmaCat No D5648;
Hanks：Gibco?Cat?No?61200-036；
Murine leukemia supressor (mLIF): SigmaCat No L5158;
Prostatropin (bFGF): Gibco Cat No 13256-029;
Human stem cell growth (hSCF): Sigma Cat No S7901;
Insulin-like growth factor-1 (IGF-I): Sigma Cat No I3769;
Foetal calf serum (FBS): Gibco Cat No 16141-061;
PAS staining fluid: Sigma Cat No 395-B;
Chicken serum (CBS): Gibco Cat No 16110-082;
Trypsin Trypsin): Gibco Cat No27250-034;
Mitomycin-C: Sigma Cat No M0503;
Beta-mercaptoethanol: Gibco Cat No 21985-023.
(3) solution
1, the preparation of PBS
NaCl8.0g, Na
2HPO
412H
2O 1.44g, KCl 0.25g, KH
2PO
40.25g, add an amount of ultrapure water and make its dissolving, with 1000ml volumetric flask constant volume.Regulate pH value about 7.2, the packing of 100ml/ bottle.15 pounds of autoclaving 30min, 4 ℃ of preservations standby (with preceding every bottle penicillin/streptomycin solution that adds a pipe step 13).
2, the preparation of penicillin/streptomycin solution (100 times of concentration)
Penicillin 1.0 * 10
6IU, Streptomycin sulphate 1g add PBS to 10ml, 0.22 μ m filtering with microporous membrane degerming.100ul/ manages packing ,-20 ℃ of preservations.
3, the preparation of Hanks liquid
Get Hanks one bag (9.8 gram) and, take by weighing NaHCO with the ultrapure water dissolving
30.35g, be dissolved in mixing among the Hanks, be settled to 1000ml with ultrapure water.The HCl of 1M transfers about pH7.2,0.22 μ m filtering with microporous membrane degerming, the packing of 100ml/ bottle.4 ℃ of preservations standby (with preceding every bottle penicillin/streptomycin solution that adds a pipe step 2).
4, the packing of Mitomycin-C
The Mitomycin-C of getting a packing adds 4ml PBS dissolves it fully, 200 μ l packing after the 0.22 μ m filtering with microporous membrane degerming, and-20 ℃ of sealings are preserved.Be ultimate density 10 μ g/ml with high sugared DMEM dilution when using.
5, the preparation of 0.1% gelatin
Take by weighing the 0.1g gelatin and fully be dissolved in the 100ml ultrapure water, 15 pounds of autoclavings, 30min.The 5ml packing, 4 ℃ of preservations are standby.
6, the making of chick embryo extract
1) ovum gallinaceum of normal fertilization is hatched in 38 ℃ of incubators or thermostat container.Put a water reservoir in the incubator and keep with the humid air in the case.Stir ovum gallinaceum 1 or 2 times every day.
2) ovum gallinaceum that will hatch 9～12 days was dipped in 95% alcohol 10～15 minutes, take out in the rearmounted small beaker, the air chamber end up, with the eggshell of the tincture of iodine and cotton ball soaked in alcohol sterilization air chamber end.
3) break the eggshell of air chamber end into pieces, peel off eggshell, carefully peel off air bag and chorioallantoic membrane, clamp chicken embryo neck, take out the chicken embryo gently, place plate with the elbow tweezers with tweezers.
4) remove chicken embryo eyes, clot and yolk, with the flushing of Hanks liquid for several times.
5) whole chicken embryo is thoroughly shredded, place tissue homogenizer to grind, or insert in the syringe, pressurization is clamp-oned tissue juice in the centrifuge tube.
6) add the Hanks liquid of organizing equivalent with the chicken embryo, blow and beat mixing gently with suction pipe, sealing was placed in 37 ℃ of thermostat containers 30 minutes, treated the nutritive ingredient leaching in the chicken embryo tissue.
7) the centrifugal 30min of 3000r/min with the packing of sterilization vial, seals.In-20 ℃ of refrigerators, preserve.
8) dilute with ultrapure water 1:1 with preceding, prepare with 1% concentration (volume ratio) during use.
7, the preparation of Digestive system
Take by weighing trypsinase 0.25g, disodium ethylene diamine tetraacetate (EDTA) 0.040g is dissolved among the 100ml PBS, places 37 ℃ of water-baths that it is dissolved fully, with 0.22 μ m filtering with microporous membrane degerming, 1ml/ manages packing, 4 ℃ of preservations are standby.
9, the preparation of inoblast nutrient solution
The high sugared DMEM:4.5g/L glucose, 10% new-born calf serum (NCS) (volume ratio), 2mM L-glutaminate, 100IU/ml penicillin and the 100 μ g/ml Streptomycin sulphates that contain following solute.
10, the preparation of the embryonic genital cell nutrient solution of chicken
The embryonic genital cell nutrient solution of chicken is high sugared DMEM, contains following solute: 4.5g/L glucose, 2mM L-glutaminate, 5.5 * 10
-5M beta-mercaptoethanol, 1mM hydroxyethyl croak piperazine second thiosulfonic acid, 100IU/ml penicillin, 100ug/ml Streptomycin sulphate, 100IU/ml leukaemia inhibitory factor, 5ng/ml stem cell factor, 10ng/ml Prostatropin, 10ng/ml insulin-like growth factor-1,10% foetal calf serum (volume ratio), 2% chicken serum (volume ratio), 1% (volume ratio) chick embryo extract.
(4) preparation of chick embryo fibroblast (PCEFs)
1, the processing of chicken embryo
1. get the chicken embryo of 9～11 ages in days, the air chamber place is with the tincture of iodine and alcohol disinfecting (smearing by around the mediad).
2. remove air chamber place's chorion and shell membrane with tweezers, take out the chicken embryo and place aseptic plate (plate is put into Hanks liquid in advance), with four limbs, head, tail and the internal organ etc. of eye scissors removal chicken embryo, 2～3 times (on Bechtop) of Hanks liquid flushing.
3. the tissue after washing is cut into 1mm with eye scissors
3Small tissue blocks, Hanks liquid flushing 2～3 times is removed hemocyte, coloring matter and cell debris etc.
2, the digestion of cell
1. 4～5 times to the Digestive system of cultured tissue volume, place 37 ℃ of digestion 15min, but the centre jog is several times, finds that tissue block becomes loose, surperficial when scared explanation digestion suitable.
2. Digestive system is poured out, and adds high sugared DMEM5～10ml after Hanks liquid cleans 2～3 times, with big suction pipe piping and druming, to break up tissue block.
3. the cell after discrete is with 2～4 layers of filtered through gauze, collecting cell suspension.
3, the cultivation of cell
1. the cell suspension that takes a morsel, trypan blue dyeing back cell counting.
2. be 2～5 * 10 with high sugared DMEM diluting cells suspension to final cell density
5Individual/ml, place culturing bottle to cultivate.
3. 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.
4. change liquid after 24 hours, remove cell lump and dead cell, went down to posterity once in 2～3 days.
4, cell goes down to posterity
1. in the time of bottom the inoblast confluent culture bottle, can go down to posterity.
2. clean 2～3 times with Hanks liquid after removing high sugared DMEM nutrient solution.
3. add Hanks liquid 2～3ml, Digestive system 200～300 μ l, 37 ℃ hatch 2～3min after, inhale and to remove Digestive system.
4. use among high sugared DMEM5～6ml and remaining trypsinase, with big suction pipe piping and druming cell dispersion.
5. collecting cell suspension, the centrifugal 8～10min of 1000rpm removes supernatant.
6. high sugared DMEM re-suspended cell group also carries out cell counting, and adjusting cell density is 2～5 * 10
5About individual/ml, place culturing bottle to cultivate.
7. 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.
(5) preparation of chick embryo fibroblast (PCEFs) feeder layer
1, in advance more than 2 hours the gelatin solution 0.1% be laid in the culture plate, suction goes gelatin, PBS solution to wash 2～3 times before using.
2, choose well-grown chick embryo fibroblast that reached for 3～6 generations inhale remove nutrient solution after, PBS washes twice, adds the Mitomycin-C solution for preparing in advance, hatches 2～2.5 hours for 37 ℃.
3, pour out Mitomycin-C solution and wash 7 times, remove fully to guarantee Mitomycin-C with PBS.Add PBS2～3ml, Digestive system 200～300ul, 37 ℃ hatch 2～3min after, inhale and remove Digestive system.
4, add high sugared DMEM5～6ml, make it be separated into single cell suspension with big suction pipe piping and druming.
5, collecting cell suspension, the centrifugal 8～10min of 1000rpm removes supernatant.
6, high sugared DMEM re-suspended cell is rolled into a ball and is carried out cell counting, and adjusting cell density is 3.5 * 10
5Individual/ml, be inoculated in 24 well culture plates.
7,37 ℃, 5% CO
2, cultivate under the saturated humidity condition.
8, observation of cell growing state after 24 hours is rarely then added cell after the processing to guarantee that cell can join together and very close to each other if find that cell is crossed.
The feeder layer for preparing is placed in the incubator standby, in 5 days, uses, be replaced by the EG cell culture fluid before the use.
The cultivation of embodiment 1, EG cell
1, takes out the overdue ovum gallinaceum of hatching (chicken deceive in Shouguang) 30 pieces, make the air chamber end upwards, the usefulness tincture of iodine and alcohol disinfecting eggshell.
2, wipe out air chamber end eggshell with sterilization, carefully push chorioallantoic membrane aside, reach idiosome neck below, provoke the chicken embryo gently and place plate with curved glass stick or elbow tweezers.
3, remove things such as embryo eye, clot, chorioallantoic membrane, with PBS flushing chicken embryo.
4, the chicken embryo is placed the plate that fills PBS, carefully push chicken embryo belly aside, remove the internal organ in the abdomen, expose middle kidney and sexual gland.With glass needle picking sexual gland and surrounding tissue thereof, Hanks liquid washes 2-3 time under the stereoscopic microscope mirror.
5, Hanks liquid is poured out Digestive system digestion 1～2min (37 ℃).
6, digestion finish, in and pancreatin, dispel cell with the rifle head, the centrifugal 5～8min of 200g.
7, separate PGCs that obtains and the small org agglomerate that contains PGCs after digestion, the centrifugal 5min of 200g.
8, immigration on 24 well culture plates of 0.01% gelatin bag quilt, adds 3～4 parts of sexual gland (estimating by the gonadal number of being got) in advance on each plate.
9, the embryonic genital cell nutrient solution that adds fresh chicken.
10, the CO that keeps 37 ℃ of humidity 90%, temperature
2Hatch in the incubator, change liquid, until more typical cell clone (about 7-10 days) occurring every one and half amounts.
11, choose cell clone, carefully blow and beat with suction pipe, 0.25% Digestive system digestion 30sec stops digestion.
12, the centrifugal 8～10min of 1000rpm moves on the fresh culture plate that is covered with PCEFs cell feeder layer and cultivates.
The evaluation of embodiment 2, EG cell
The EG cell that embodiment 1 is cultivated reached for the 4th generation, carried out following evaluation:
One, morphology is identified
The EG cell clone is the island or the growth of colony shape of rule, and multilayer distributes, and tangible decorative pattern is arranged, and iuntercellular is not closely arranged, and can see inner cell boundary sometimes clearly.Class EG cell contains big nucleus and relative less kytoplasm, and kernel is not obvious.
Two, PAS staining reaction:
Fixedly behind the 5min, about 5min in the periodic acid reagent is immersed in 1 * PBS flushing 2 times to the EG cell under the room temperature in 1% glutaraldehyde, use 1 * PBS flushing again, then immerse about 15min in the Schiff's reagent, 1 * PBS flushing 2 times is observed under inverted microscope through the painted EG cell of PAS.
EG cell colony and single PGCs and be amaranth show positive; Chick embryo fibroblast is not painted.
Three, vitro differentiation experiment
1, spontaneous differentiation
Accumulative EG cell colony is removed the somatomedin in the nutrient solution, does not change feeder layer, does not go down to posterity for a long time, observes the colony form of its spontaneous differentiation.
The visible cell poor growth, spontaneous differentiation at first shows as and loses original form, and it is big that cell volume becomes, and then unclear with the peripheral cell boundary, and the cell and the feeder layer of colony periphery join together.
2, suspension culture
In order to identify whether the EG cell can form embryoid, choose cell clone after suction pipe is centrifugal after carefully blowing and beating.The EG cell the EG of no mLIF substratum (the EG culture medium prescription see embodiment 1 step 1 (three) 10) in resuspended cultivation, nutrient solution is every other day changed once.Every day, microscopy was observed the situation that EG cytodifferentiation and embryoid form.
After 3～4 days, the visible part cell mass forms simple idiosome, and outermost layer is divided into by the entoderm spline structure of forming than maxicell, and the center is undifferentiated cell, similar body early embryo.
Four, the making of mosaic chicken
1. the chicken EG cell that reaches 3 generations, 4 generations of Pei Yanging is a donor.At first select cell clone, PBS cleans the back and digests 2～3min for 37 ℃ with Digestive system.
2. the EG cell clone is moved into and wash twice back among the high sugared DMEM and move among the PBS.
3. blow and beat gently with a mouthful suction pipe and by the effect of peeling off pin cell mass is separated into single cell suspension, the centrifugal 8～10min of 1000rpm removes supernatant, collecting cell group.
4. use PBS re-suspended cell group, the cell suspension of making cell density and be 100/ul is standby.
5. be acceptor (45 pieces) with the blue white race egg in sea, donorcells is injected in the subgerminal cavity of acceptor kind egg, change over to and substitute among the eggshell I, add egg white (two anti-final concentrations are penicillin 250IU/ml, Streptomycin sulphate 250ug/ml) to full, preservative film sealed (38.2 ℃, RH50%, 90 ° of egg-turning angles) hatching to 72 hours.
6. change and substitute among the eggshell II (38.2 ℃, RH60%, 30 ° of egg-turning angles) hatching then over to until hatching in 21 days.Survive 4, youngly a little less than having one be the hair color mosaic.
Claims (6)
1, the embryonic genital cell nutrient solution of chicken, it is high sugared DMEM solution, contains following solute: 3.5-4.5g/L glucose, 1.5-2.5mM L-glutaminate, (4.5-5.5) * 10
-5M beta-mercaptoethanol, 1-1.5mM hydroxyethyl croak piperazine second thiosulfonic acid, 100-120IU/ml leukaemia inhibitory factor, 4-6ng/ml stem cell factor, 10-12ng/ml Prostatropin, 10-12ng/ml insulin-like growth factor-1,10-15% foetal calf serum (volume ratio), 1-3% chicken serum (volume ratio), 1-1.2% chick embryo extract (volume ratio), antibiotic.
2, nutrient solution as claimed in claim 1 is characterized in that: described antibiotic is penicillin and Streptomycin sulphate; Contain 100-150IU/ml penicillin, 100-120 μ g/ml Streptomycin sulphate in the described nutrient solution.
3, nutrient solution as claimed in claim 1 or 2 is characterized in that nutrient solution contains following solute: 4.5g/L glucose, 2mM L-glutaminate, 5.5 * 10
-5M beta-mercaptoethanol, 1mM hydroxyethyl croak piperazine second thiosulfonic acid, 100IU/ml penicillin, 100ug/ml Streptomycin sulphate, 100IU/ml leukaemia inhibitory factor, 5ng/ml stem cell factor, 10ng/ml Prostatropin, 10ng/ml insulin-like growth factor-1,10% foetal calf serum (volume ratio), 2% chicken serum (volume ratio), 1% (volume ratio) chick embryo extract.
4, a kind of method of cultivating the embryonic genital cell of chicken is that embryonic genital cell with chicken places on the chick embryo fibroblast culture layer and cultivates.
5, method as claimed in claim 4 is characterized in that: the embryonic genital cell of described chicken is that the embryonic germ gland primordial germ cells cell of the chicken that will be separated to and chicken embryo sexual gland add to cultivate in arbitrary described cell culture fluid in the claim 1 to 3 and obtains.
6, as claim 4 or 5 described methods, it is characterized in that: described chicken embryo PGCs cell is the 5.5-8.5 embryo cell in age.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384749A (en) * | 2017-12-07 | 2018-08-10 | 广西大学 | Chicken sexual gland archaeocyte quick separating and build the method for being |
| CN111793652A (en) * | 2020-07-14 | 2020-10-20 | 扬州大学 | Method for efficiently constructing chimera gonad chick embryo through embryo transfer technology |
| CN114410572A (en) * | 2022-01-24 | 2022-04-29 | 成都思澍生物科技有限公司 | Method for quickly separating high-purity primordial germ cells from chick embryo blood |
-
2008
- 2008-12-18 CN CNA2008102400944A patent/CN101423818A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384749A (en) * | 2017-12-07 | 2018-08-10 | 广西大学 | Chicken sexual gland archaeocyte quick separating and build the method for being |
| CN108384749B (en) * | 2017-12-07 | 2021-08-17 | 广西大学 | Method for quickly separating and establishing chicken gonad primordial germ cells |
| CN111793652A (en) * | 2020-07-14 | 2020-10-20 | 扬州大学 | Method for efficiently constructing chimera gonad chick embryo through embryo transfer technology |
| CN114410572A (en) * | 2022-01-24 | 2022-04-29 | 成都思澍生物科技有限公司 | Method for quickly separating high-purity primordial germ cells from chick embryo blood |
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