CN112553140B - In-vitro amplification culture system for primordial germ cells of female chickens and application of in-vitro amplification culture system - Google Patents
In-vitro amplification culture system for primordial germ cells of female chickens and application of in-vitro amplification culture system Download PDFInfo
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Abstract
The invention discloses an in-vitro amplification culture system for primordial germ cells of female chickens and application thereof. The culture system of the invention comprises the following components: 52-54% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture fluid, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) nonessential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 1-3% (v/v) aromatase culture fluid, 4ng/mL SCFRMm, 2.5ng/mL rhFGF. The success rate of the culture system for amplifying the primordial germ cells of the female chicken can reach 37.5 percent at most, and the undifferentiated state and the good biological characteristics of the primordial germ cells of the female chicken can be maintained.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an in-vitro amplification culture system for female chicken primordial germ cells and application thereof.
Background
The main role of poultry resource conservation is living organism conservation, but the costs for raising, placing, and labor of living organism conservation are high, and most of the conservation groups are temporarily unavailable groups, so that other conservation techniques need to be developed. Primordial Germ Cells (PGCs) originate in the epiblast and are early cells that eventually form male or female germ cells and can transfer genes from the previous generation to the next. The separation, culture and preservation of primordial germ cells are expected to realize the function of in vitro conservation of poultry resources, and if the primordial germ cells can be subjected to gene manipulation, the sperms or ova generated by transplantation can be used for producing transgenic or gene-edited animals. At present, the in vitro culture system of chicken primordial germ cells mainly comprises the following components: KO-DMEM, Fetal Bovine Serum (FBS), chicken serum, nonessential amino acids, GlutaMAXTM, pyruvic acid, beta-mercaptoethanol, recombinant human basic fibroblast growth factor (rhFGF), human leukemia inhibitory factor (human LIF), or human stem cell factor (human SCF). By utilizing the systems, the success rate of separating and culturing the primordial germ cells of the male chicken exceeds 50 percent, but the success rate of separating and culturing the primordial germ cells of the female chicken is only about 3 percent, even lower. Therefore, the in vitro culture system is more suitable for the primordial germ cells of male chickens and is not suitable for the primordial germ cells of female chickens.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an in-vitro amplification culture system for primordial germ cells of female chickens.
The invention also aims to provide the application of the in-vitro amplification culture system for the primordial germ cells of the female chicken.
The purpose of the invention is realized by the following technical scheme: an in-vitro amplification culture system for female chicken primordial germ cells comprises the following components: KO-DMEM with the final concentration of 52-54% by volume is taken as a basic culture medium, and the following components are added in proportion: rat hepatocyte culture fluid with the final concentration of 30% by volume, Fetal Bovine Serum (FBS) with the final concentration of 7.5% by volume, chicken serum with the final concentration of 2.5% by volume, nonessential amino acid with the final concentration of 1% by volume, GlutaMAXTM culture medium with the final concentration of 1% by volume, pyruvic acid with the final concentration of 1% by volume, beta-mercaptoethanol with the final concentration of 1% by volume, penicillin streptomycin with the final concentration of 1% by volume, aromatase culture fluid with the final concentration of 1-3% by volume, recombinant mouse stem cell factor (rmSCF) with the final concentration of 4ng/mL, and recombinant human basic fibroblast growth factor (rhFGF) with the final concentration of 2.5 ng/mL.
Preferably, the in vitro amplification culture system for the female chicken primordial germ cells comprises the following components: KO-DMEM with the final concentration of 52% by volume is taken as a basic culture medium, and the following components are added in proportion: rat hepatocyte culture fluid with the final concentration of 30 percent by volume, fetal bovine serum with the final concentration of 7.5 percent by volume, chicken serum with the final concentration of 2.5 percent by volume, nonessential amino acid with the final concentration of 1 percent by volume, GlutaMAXTM culture medium with the final concentration of 1 percent by volume, pyruvic acid with the final concentration of 1 percent by volume, beta-mercaptoethanol with the final concentration of 1 percent by volume, penicillin streptomycin with the final concentration of 1 percent by volume, aromatase culture fluid with the final concentration of 3 percent by volume, rmSCF with the final concentration of 4ng/mL and rhFGF with the final concentration of 2.5 ng/mL.
The preparation of the aromatizing enzyme culture solution preferably comprises the following steps: amplifying an aromatase gene CYP19A1 by using gonad cDNA as a template, constructing a recombinant vector, converting, selecting positive clones to extract plasmid p-EGFP-CYP, transfecting the plasmid p-EGFP-CYP in DF1 cells, screening the cells stably transfected with the plasmid p-EGFP-CYP, carrying out amplification culture, and collecting supernatant to obtain an aromatase culture solution.
The primers for amplifying the aromatizing enzyme gene CYP19A1 are as follows: an upstream primer 5'-AGAAAACCTACTCAGA-3' and a downstream primer 5'-GGCTTACAATAATGGTAG-3'.
The vector adopted for constructing the recombinant vector is p-EGFP.
The transformation described was transformed E.coli DH5 alpha.
The DF1 cells were cultured at 37 ℃ with 5% CO 2 DF1 cells growing to the cell density of 80-90% under the condition; the culture system comprises 90% KO-DMEM by volume and 10% fetal bovine serum by volume.
The transfection is preferably every 10 th 8 Mu.g of plasmid p-EGFP-CYP was transfected into each DF1 cell, and the cells were changed 6 hours later.
The supernatant is obtained once every 3 days, and is mixed.
The preparation of the rat hepatocyte culture solution preferably comprises the following steps: resuscitating rat liver cells at 37 deg.C and 5% CO 2 Culturing for 9 days under the condition, collecting supernatant liquid once after 3 days, and mixing to obtain the rat hepatocyte culture solution.
The culture system comprises: 89% by volume KO-DMEM, 10% by volume fetal bovine serum and 1% by volume GlutaMAXTM.
The application of the in-vitro amplification culture system of the female chicken primordial germ cells in the large-scale culture of the female chicken primordial germ cells.
The application is that the female chicken primordial germ cells are inoculated into a female chicken primordial germ cell in-vitro amplification culture system and a rat liver cell feeder layer and cultured to obtain the female chicken primordial germ cells cultured in a large scale.
The culture is carried out at 37 ℃ and 5% CO 2 Culturing under the condition; half of the cells were changed 1 time every 2 days, and the cells were passaged 1 time every 4-5 days.
The preparation of the rat hepatocyte feeder layer preferably comprises the following steps: resuscitating rat liver cells at 37 deg.C and 5% CO 2 Culturing under the condition until the cell density is more than 90%, digesting the cells with 2mL of pancreatin with the concentration of 0.01ng/mL for 5min, centrifuging at 1000g/min for 5min, collecting the cells, and radiating with a cobalt source at 50gray/min for 12min to obtain a rat hepatocyte feeder layer.
Compared with the prior art, the invention has the following advantages and effects:
1. the existing in-vitro culture system of the chicken primordial germ cells is more suitable for the proliferation and separation culture of the male chicken primordial germ cells, the success rate of the in-vitro culture system of the female chicken primordial germ cells is only about 3 percent, even lower, the in-vitro culture system of the female chicken primordial germ cells can efficiently amplify the female chicken primordial germ cells in vitro, the success rate reaches 37.5 percent, and the undifferentiated state and good biological characteristics of the female chicken primordial germ cells can be maintained.
Drawings
FIG. 1 is a photograph (100X) of primordial germ cells of a female chicken cultured for 10 days in the culture system (c) of example 1.
FIG. 2 is a photograph (100X) of a female chicken primordial germ cell cultured in the culture system (R) of example 1 for 10 days.
FIG. 3 is a graph showing the effect of different amounts of aromatase culture medium on the proliferation of primordial germ cells of female chickens in culture System (r) of example 1.
FIG. 4 is the electrophoresis chart of sex determination of primordial germ cells of female chicken obtained by isolated culture in example 2.
FIG. 5 is the expression pattern of female chicken primordial germ cell surface marker protein SSEA-1 obtained by isolated culture in example 2.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Embodiments of the present invention will be described in detail below with reference to embodiments and the accompanying drawings, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In examples 1 and 2 chicken serum, non-essential amino acids, GlutaMAXTM medium and pyruvate were purchased from Invitrogen, beta-mercaptoethanol was purchased from sigma, and rmSCF and rhFGF were purchased from R & D systems.
Example 1
(1) Preparation of aromatase culture solution
Searching sequence (ENSGALG00000013294) of aromatizing enzyme gene CYP19A1 in an ENSEMBL website, designing a primer (upstream: 5'-AGAAAACCTACTCAGA-3', downstream: 5'-GGCTTACAATAATGGTAG-3'), and amplifying the gene segment by taking gonad cDNA as a template. A vector with a target DNA was constructed using an In-Fusion Kit (In-Fusion HD Cloning Kit, TaKaRa Co.), Escherichia coli DH5 alpha (TaKaRa Co.) was transformed, a positive clone was selected, sequencing was performed to determine the sequence, and then amplification culture was performed, and a positive clone Plasmid p-EGFP-CYP was extracted using an endotoxin-free Plasmid mimi Kit (OMEGA). Resuscitating DF1 cells (
CRL-12203 TM ATCC Co.) to 10cm cell culture dish (Thermo Co.), 37 ℃ 5% CO 2 Culturing under the condition (the culture system is KO-DMEM (Gibco) with the volume ratio of 90 percent, fetal bovine serum (Gibco) with the volume ratio of 10 percent, when the cell grows to the cell density of 80-90 percent, transfecting plasmid p-EGFP-CYP (10 percent) by using a lip2000 liposome transfection kit (Invitrogen) 8 Transfecting 2 mu g of plasmid into DF1 cells, changing the liquid after 6 hours, continuously culturing for 6 days, then sorting by a flow cytometer to obtain positive cells stably transfected with p-EGFP-CYP plasmid, and freezing and storing after amplification culture. Resuscitating positive cells to 10cm cell culture dish, 5% CO at 37 deg.C 2 Culturing under the condition (the culture system is KO-DMEM (Gibco) with the volume ratio of 90% and fetal bovine serum (Gibco) with the volume ratio of 10%), changing the culture solution when the cell grows to the cell density of 80-90%, continuously culturing for 9 days, collecting the culture solution once every 3 days, and mixing the supernatant to obtain the aromatizing enzyme culture solution.
(2) Preparation of rat hepatocyte culture solution
Resuscitating rat liver cells (
CRL-1442 TM ATCC Co.) into 10cm cell culture dishes at 37 ℃ with 5% CO 2 Culturing in an incubator for 9 days under the condition, and collecting liquid once every 3 days, wherein the culture system is as follows: 89% by volume KO-DMEM, 10% by volume fetal bovine serum and 1% by volume GlutaMAXTM. And mixing the collected supernatants to obtain the rat liver cell culture solution.
(3) Preparation of feeder layer rat hepatocytes
Resuscitating rat liver cells
CRL-1442 TM ATCC Co.) into 10cm cell culture dishes at 37 ℃ with 5% CO 2 Culturing in incubator under the condition until cell density is above 90%, digesting with 2mL pancreatin (Invitrogen) with concentration of 0.01ng/mL for 5min, centrifuging at 1000g/min for 5min, collecting cells, irradiating with cobalt source at 50gray/min for 12min to obtain large feeder layerMurine hepatocytes.
(4) Isolation culture of female chicken primordial germ cells
The hatching egg is Huiyang beard chicken and comes from the original breeding field of animal science research institute of agriculture academy of sciences of Guangdong province. The chick embryos are incubated at 37.8 ℃ and 60% relative humidity. Taking chicken embryo limbs which are hatched for 5-7 days, extracting DNA for sex identification, simultaneously separating chicken primordial germ cells from gonad tissues, inoculating the female chicken primordial germ cells to a female chicken primordial germ cell culture system and feeder layer rat liver cells treated by a radioactive source, and culturing at 37 ℃ with 5% CO 2 Culturing in an incubator under the condition, changing the liquid for 1 time in half every 2 days, and carrying out passage for 1 time every 4-5 days. The culture system of the female chicken primordial germ cells is as follows: 49-54% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture medium, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) nonessential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 1-6% (v/v) aromatase culture medium, 6ng/mL rmSCF, 4ng/mL rhFGF; 49-54% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture fluid, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) non-essential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 1-6% (v/v) aromatase culture fluid, 3ng/mL rmSCF, 2ng/mL rhFGF; ③ 49-51% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture medium, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) nonessential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 4-6% (v/v) aromatase culture medium, 4ng/mL rmSCF, 2.5ng/mL rhFGF; 52-54% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture medium, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) nonessential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 1-3% (v/v) aromatase culture medium, 4ng/mL rmSCF, 2.5ng/mL rhFGF. 55% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture medium, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum1% (v/v) non-essential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvic acid, 1% (v/v) β -mercaptoethanol, 1% (v/v) penicillin streptomycin, 6ng/mL rmSCF, 4ng/mL rhFGF; wherein, the fifth is a male chicken primordial germ cell culture system with the success rate of 50 percent. The cultivation system (first-fifth) is set with 6, 3, 1 groups of experiments, each group of experiments is repeated for 3 times. Except the culture system, all the experimental settings of the rest culture systems are gradient experiments, namely the volume percentage of KO-DMEM in the range of the culture system is sequentially reduced by 1 percent, and the volume percentage of the aromatase culture solution is sequentially increased by 1 percent.
The result shows that compared with a culture system of the primordial germ cells of the male chicken, 1-6% of aromatizing enzyme culture solution is added in the culture system I, the dosage of KO-DMEM is correspondingly reduced, the culture is carried out for about 7 days, and the culture result shows that all primordial germ cells of 6 experiments are dead; then, in the second culture system, the dosage of the rmSCF and the rhFGF is adjusted to be half of that in the first culture system, the culture is carried out for about 7 days, and all primordial germ cells in the 6 experiments are dead; the usage amounts of the rmSCF and the rhFGF in a culture system (three) are respectively 4ng/mL and 2.5ng/mL, after a system with 4-6% of aromatizing enzyme culture solution added is cultured for 10 days, the growth state of the primordial germ cells of the female chickens of 3 experiments is poor, and the primordial germ cells die successively (the growth state of the primordial germ cells of the female chickens cultured for 10 days is shown in figure 1 by the culture system (three) with 49% of KO-DMEM and 6% of aromatizing enzyme culture solution in volume percent), but compared with the culture systems (i) and (ii), the culture time of the primordial germ cells is longer, and white arrows in figure 1 show the dead primordial germ cells; compared with the culture system ③, the proportion of the added culture solution of the aromatizing enzyme is 1-3%, after 10 days of culture, the primordial germ cells of the female chickens of 3 groups of experiments are round, have bright edges, large volume and large cell nucleus, and the two cells are usually seen to be connected together or form a string, which accords with the morphological characteristics of the primordial germ cells of the female chickens, so that the culture system is helpful for the in-vitro proliferation of the primordial germ cells of the female chickens (figure 2 shows that the growth state of the primordial germ cells of the female chickens cultured for 10 days under the culture system of which the KO-DMEM volume percentage is 52% and the aromatizing enzyme culture solution volume percentage is 3%). Counting the primordial germ cells of the female chicken obtained after culturing 20 days under the condition that the addition amount of the aromatase culture solution in the culture system (KO-DMEM is 54%, 53% and 52% in volume ratio respectively) is 1%, 2% and 3%, collecting the cells in a centrifuge tube, adding a buffer solution PBS (0.01M, pH 7.4.4) to make the volume be 1mL, taking 20 microliter to drop into a cell counter for counting, and obtaining the result shown in figure 3. Through one-way anova, the difference between the treatment groups with the addition amount of the aromatase culture solution of 1% and 2% by volume is not significant, and the cell number of the treatment group with the addition amount of 3% by volume is significantly higher than that of the treatment groups with the addition amount of 1% and 2% by volume (P < 0.01). The result shows that the culture effect of the primordial germ cells of the female chicken is the best when the addition amount of the aromatase culture solution in the culture system is 3 percent.
Example 2
Hatching eggs of Huiyang beard chickens (from original breeding farms of animal science research institute of agricultural academy of sciences, Guangdong province) for 5-7 days, separating chicken primordial germ cells from chicken gonad tissues, and simultaneously extracting DNA from limbs for sex identification. After sex determination, the primordial germ cells of the female chickens were inoculated onto the culture system and the feeder layer of the hepatocytes of the rats treated with the radioactive source in step (3) of example 1, at 37 ℃ with 5% CO 2 Culturing in an incubator under the condition. The culture system is as follows: 52% (v/v) KO-DMEM, 30% (v/v) rat hepatocyte culture, 7.5% (v/v) fetal bovine serum, 2.5% (v/v) chicken serum, 1% (v/v) nonessential amino acids, 1% (v/v) GlutaMAXTM, 1% (v/v) pyruvate, 1% (v/v) beta-mercaptoethanol, 1% (v/v) penicillin streptomycin, 3% (v/v) aromatase culture, 4ng/mL rmSCF, 2.5ng/mL rhFGF. 16 female chick embryos are separated in total, and after 30 days, the primordial germ cells of 6 chick embryos are transferred to a T75 cell culture bottle (Thermo), which indicates that the separation success rate of the primordial germ cells of the female chick reaches 37.5%. Continuously culturing for 3 days, collecting cells, treating a part of the cells with a HotShot lysate and a HotShot neutralizing solution (Beijing Biotechnology and technology, Inc.), performing sex identification by PCR, performing immunofluorescence assay on a part of the cells, namely preparing a cell slide in a 24-hole cell culture plate, culturing chicken primordial germ cells, cleaning, fixing, permeating and cleaning the cells after 3 days, adding protease K for sealing, and performing anti-hatchingAnd (4) after overnight incubation, cleaning the cells the next day, incubating the cells with secondary antibody, then cleaning the cells, and finally taking pictures in a laser confocal microscope. The sex determination result shows that the primordial germ cells belong to female (figure 4). The results of immunofluorescence experiments show that the germ cell surface marker protein SSEA-1(Santa Cruz company) is expressed in primordial germ cells (figure 5), and the culture system can enable the primordial germ cells of female chickens to keep the undifferentiated state and good biological characteristics.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> animal science institute of academy of agricultural sciences of Guangdong province
<120> in-vitro amplification culture system for primordial germ cells of female chicken and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<223> upstream primer
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agaaaaccta ctcaga 16
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> downstream primer
<400> 2
ggcttacaat aatggtag 18
Claims (9)
1. An in-vitro amplification culture system for primordial germ cells of female chickens is characterized by comprising the following components: KO-DMEM with the final concentration of 52-54% by volume is taken as a basic culture medium, and the following components are added in proportion: rat hepatocyte culture fluid with the final concentration of 30 percent by volume, fetal bovine serum with the final concentration of 7.5 percent by volume, chicken serum with the final concentration of 2.5 percent by volume, non-essential amino acid with the final concentration of 1 percent by volume, GlutaMAXTM culture medium with the final concentration of 1 percent by volume, pyruvic acid with the final concentration of 1 percent by volume, beta-mercaptoethanol with the final concentration of 1 percent by volume, penicillin streptomycin with the final concentration of 1 percent by volume, aromatase culture fluid with the final concentration of 1 to 3 percent by volume, rmSCF with the final concentration of 4ng/mL and rhFGF with the final concentration of 2.5 ng/mL;
the preparation of the aromatizing enzyme culture solution comprises the following steps: amplification of aromatase Gene Using gonad cDNA as templateCYP19A1Constructing a recombinant vector, transforming, selecting positive clones to extract plasmid p-EGFP-CYP, transfecting the plasmid p-EGFP-CYP in DF1 cells, screening the cells stably transfected with the plasmid p-EGFP-CYP, carrying out amplification culture, and collecting supernatant to obtain an aromatase culture solution;
the aromatizing enzyme geneCYP19A1The sequence of (a) is shown in ENSGALG 00000013294.
2. The in vitro amplification culture system for the primordial germ cells of the female chicken as claimed in claim 1, which is characterized by comprising the following components: KO-DMEM with the final concentration of 52% by volume is taken as a basic culture medium, and the following components are added in proportion: rat hepatocyte culture fluid with the final concentration of 30 percent by volume, fetal bovine serum with the final concentration of 7.5 percent by volume, chicken serum with the final concentration of 2.5 percent by volume, nonessential amino acid with the final concentration of 1 percent by volume, GlutaMAXTM culture medium with the final concentration of 1 percent by volume, pyruvic acid with the final concentration of 1 percent by volume, beta-mercaptoethanol with the final concentration of 1 percent by volume, penicillin streptomycin with the final concentration of 1 percent by volume, aromatase culture fluid with the final concentration of 3 percent by volume, rmSCF with the final concentration of 4ng/mL and rhFGF with the final concentration of 2.5 ng/mL.
3. The in vitro amplification culture system for the female chicken primordial germ cells, according to claim 1, is characterized in that:
the amplified aromatase geneCYP19A1The primer is as follows: upstream primer 5'-AGAAAACCTACTCAGA-3', downstream primer: 5'-GGCTTACAATAATGGTAG-3', respectively;
the vector adopted for constructing the recombinant vector is p-EGFP;
the transformation is transformed Escherichia coli DH5 alpha;
the DF1 cells were cultured at 37 ℃ with 5% CO 2 DF1 cells growing to the cell density of 80-90% under the condition; the culture system comprises 90% KO-DMEM by volume and 10% fetal calf serum by volume;
the transfection is every 10 8 2 mug plasmid p-EGFP-CYP is transfected in each DF1 cell, and the liquid is changed after 6 hours;
the supernatant is obtained once every 3 days, and is mixed.
4. The in vitro amplification culture system for the female chicken primordial germ cells according to claim 1 or 2, wherein:
the preparation of the rat hepatocyte culture solution comprises the following steps: resuscitating rat liver cells at 37 deg.C and 5% CO 2 Culturing for 9 days under the condition, collecting supernatant liquid once after 3 days, and mixing to obtain the rat liver cell culture solution.
5. The in vitro amplification culture system for female chicken primordial germ cells as claimed in claim 4, wherein: the culture system of rat liver cells is as follows: 89% by volume KO-DMEM, 10% by volume fetal bovine serum and 1% by volume GlutaMAXTM.
6. The application of the in-vitro amplification culture system for the female chicken primordial germ cells, disclosed by any one of claims 1 to 5, in large-scale culture of the female chicken primordial germ cells.
7. The application of the in vitro amplification culture system of the female chicken primordial germ cells in the large-scale culture of the female chicken primordial germ cells according to the claim 6, is characterized in that: inoculating the primordial germ cells of the female chicken to a primordial germ cell in-vitro amplification culture system of the female chicken and a rat liver cell feeder layer, and culturing to obtain the primordial germ cells of the female chicken cultured in a large scale.
8. The application of the in vitro amplification culture system of the female chicken primordial germ cells in the large-scale culture of the female chicken primordial germ cells according to the claim 7 is characterized in that: the preparation of the rat hepatocyte feeder layer comprises the following steps: resuscitating rat liver cells at 37 deg.C and 5% CO 2 Culturing under the condition until the cell density is more than 90%, digesting the cells with 2mL of pancreatin with the concentration of 0.01ng/mL for 5min, centrifuging for 5min at 1000g/min, collecting the cells, and radiating for 12min by a cobalt source at 50gray/min to obtain the rat hepatocyte feeder layer.
9. The application of the in vitro amplification culture system of the female chicken primordial germ cells in the large-scale culture of the female chicken primordial germ cells according to the claim 7 is characterized in that: the in-vitro amplification culture system for inoculating the female chicken primordial germ cells into the female chicken primordial germ cells and the rat liver cell feeder layer are cultured at 37 ℃ and 5 percent CO 2 Culturing under the condition; half of the cells were changed 1 time every 2 days, and the cells were passaged 1 time every 4-5 days.
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| AU2001291520A1 (en) * | 2000-10-09 | 2002-06-27 | Commonwealth Scientific And Industrial Research Organisation | Genetic control of sex ratio in animal populations |
| CN1821389A (en) * | 2006-03-02 | 2006-08-23 | 扬州大学 | External Cultivating method for bird primordial germ cell |
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