A method for establishing a line of avian female primordial germ cells

Abstract

The application discloses a method for establishing a line of female primordial germ cells of poultry, comprising the following steps: (1) sampling; placing fertilized eggs in an incubator to hatch to 5.5-7.5 days, taking out the embryo of the fertilized egg, and separating the left gonad under a stereomicroscope; and taking a small amount of tissue for gender identification; (2) separating cells; digesting and grinding the left gonad, and stopping digestion with complete culture medium after digestion; centrifuging to collect the bottom cells, and resuspending to obtain a single-sided derived primordial germ cell suspension; (3) culturing cells; inoculating the left gonad derived cells into a feeder-free culture medium, and continuously culturing to complete the line establishment. Compared with the prior primordial germ cell separation method, the application can improve the culture stability and the clone formation ability of the female primordial germ cells, is beneficial to long-term culture and expansion of the female primordial germ cells, and can be applied to the fields of genetic improvement, germplasm resource preservation and gene editing breeding of poultry.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for establishing a lineage of female primordial germ cells in poultry. Background Technology

[0002] Primordial germ cells (PGCs), as precursor cells for gamete development, can transmit genetic information to the cell lineage of offspring, and have core application value in poultry genetic improvement, germplasm resource preservation, and genome editing breeding. Chicken PGCs migrate directionally through the embryonic blood circulation and eventually colonize the genital ridge. The gonads are highly enriched when the embryo develops to 5.5–7.5 days, which is the ideal time to isolate gonadal PGCs.

[0003] Recent studies have confirmed that female avian embryos exhibit significant left-right asymmetric gonadal development, with only the left gonad differentiating into a functional ovary and enriching with highly active germ cells, while the right gonad degenerates. Compared to male progenitor cells (PGCs), female PGCs have lower self-renewal potential and are more difficult to culture in vitro, exhibiting slower proliferation rates and a tendency to aggregate and undergo apoptosis, resulting in lower establishment efficiency for female PGCs compared to males.

[0004] Existing methods for isolating gonads from PGCs suffer from problems such as low efficiency in establishing female PGC lines, poor culture stability, and insufficient cell viability, which severely restrict the large-scale application of female PGCs in poultry reproductive engineering, germplasm preservation, and gene editing breeding.

[0005] Therefore, establishing a suitable method for the stable establishment of female PGCs is of great practical significance for promoting the research and application of female primordial germ cells in poultry. Summary of the Invention

[0006] Purpose of the invention: In order to overcome the low efficiency and insufficient cell viability of existing poultry female primordial germ cell isolation and establishment methods, this invention provides a method for establishing poultry female primordial germ cells. By culturing them separately on the left side, the establishment efficiency and cell viability are significantly improved. This method can be used for poultry reproductive engineering, genetic improvement, germplasm resource preservation and developmental biology research.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a method for establishing a lineage of female primordial germ cells in poultry, comprising the following steps:

[0008] (1) Sampling; fertilized eggs are placed in an incubator and incubated for 5.5 to 7.5 days. The embryos of the fertilized eggs are removed and the left gonad is separated under a stereomicroscope. A small amount of tissue is taken for sex identification.

[0009] (2) Cell separation; digest and grind the left gonad, and stop digestion with complete culture medium after digestion; collect the bottom cells by centrifugation, and resuspend to obtain a suspension of primordial germ cells from the left gonad;

[0010] (3) Cell culture: The cells derived from the left gonad were seeded in a culture medium without a feeder layer and cultured continuously to complete the establishment of the cell line.

[0011] In step (1), when the eggshell is incubated for 5.5 to 7.5 days, the eggshell surface is disinfected with alcohol, the eggshell is opened and the chicken embryo is taken out. The embryo is placed in a dish containing PBS, the blood on the embryo is washed off, the left gonad is found under a stereomicroscope, it is separated, and a 0.5×0.5 mm tissue is taken with fine tweezers and placed in a PCR tube.

[0012] In step (1), the sex determination is performed using the direct PCR amplification method.

[0013] In step (2), the complete culture medium is DMEM medium containing 10% FBS.

[0014] In step (3), the feederless culture medium consists of KO DMEM basal medium (KnockOut). TM DMEM, Thermo Scientific TM The following ingredients were added: B-27, GlutaMAX, MEM non-essential amino acid solution, β-mercaptoethanol, nucleoside, sodium pyruvate, ovalbumin, sodium heparin, Human activin A, FGF2, chicken serum and bispecific antibodies.

[0015] The continuous culture mentioned in step (3) refers to continuous culture under the conditions of 37℃, 5% CO2, and 60~70% relative humidity.

[0016] In step (3), during the continuous culture process, half of the culture medium is replaced every two days. When replacing the medium, the well plate containing the culture medium is tilted, the old culture medium is taken from the top layer of the liquid, and then preheated fresh culture medium without feeder layer is added. The pipette tip is replaced each time the medium is changed to avoid cross-contamination. When too many adherent cells affect the growth of primordial germ cells, the adherent somatic cells at the bottom of the culture plate are digested with Accutase enzyme to passage the primordial germ cells.

[0017] In step (3), during the continuous culture process, when obvious clones of the primordial germ cells appear, they are passaged or transferred to culture plates with larger pore sizes for amplification. When the cell number reaches 1×10⁻⁶, the culture continues. 5 The cell line was determined to be successfully established at this time.

[0018] Preferably, the method for establishing a lineage of female primordial germ cells in poultry according to the present invention includes the following steps:

[0019] (1) Sampling; Fertilized eggs are placed in an incubator and incubated for 5.5 to 7.5 days. The eggshell surface is wiped with 75% alcohol for disinfection. The eggshell is opened and the embryo of the fertilized egg is taken out. The left gonad is separated under a stereomicroscope. At the same time, a tissue of 0.5 × 0.5 mm is taken for sex identification.

[0020] (2) Cell isolation; add 0.25% trypsin for digestion, grind with a grinding stick, and terminate digestion with DMEM complete medium containing 10% FBS after digestion. Centrifuge at 1500 rpm for 5 min to collect the bottom cells, and resuspend to obtain a suspension of primordial germ cells from one side;

[0021] (3) Cell culture: Left gonadal-derived cells were seeded in a feeder-free culture medium and cultured continuously at 37°C, 5% CO2, and 60-70% relative humidity to establish the cell line. When too many adherent cells affected the growth of primordial germ cells, the primordial germ cell suspension was collected, and the adherent cells at the bottom of the culture plate were digested with Accutase enzyme to passage the primordial germ cells.

[0022] In step (1), fresh fertilized eggs are incubated in an incubator (temperature 37-38℃, relative humidity 58%, eggs are turned every 30 minutes at an angle of 45°) and taken out after 5.5-7.5 days.

[0023] Further, in step (1), the egg is cut open, the chicken embryo is taken out, and it is washed in a culture dish containing PBS solution. Then, under a stereomicroscope, the left gonad of the chicken embryo is carefully peeled off and separated, and a 0.5×0.5 mm tissue is taken into a PCR tube with fine tweezers.

[0024] Furthermore, in step (1), during sex identification, the obtained tissue is used as a template to directly perform PCR amplification, and the amplification products are detected by gel electrophoresis. The sex of the chicken embryo is identified by the size of the amplification products.

[0025] In step (2), after briefly separating the centrifuge tube containing gonadal tissue and discarding PBS, 0.25% trypsin was added for digestion for 1 min. During digestion, a sterile grinding rod was used to grind the cells while digesting. After digestion, DMEM medium containing 10% FBS was added to terminate the digestion. The cell suspension was collected, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in KO-DMEM basal medium without a feeder layer. The following components were added: 1×B-27, 2 mM GlutaMAX, 1× non-essential amino acid solution, 0.1 mM β-mercaptoethanol, 1× nucleoside, 1.2 mM sodium pyruvate, 0.2% ovalbumin, 0.2% sodium heparin, 25 ng / ml human activin A, 4 ng / ml FGF2, 0.2% chicken serum, and 1% penicillin-dextrin antibiotics.

[0026] In step (3), half of the culture medium is replaced every two days during the culture process. When replacing the medium, the plate containing the culture medium is tilted, the old culture medium is taken out from the top layer of the liquid, and then preheated fresh culture medium without feed layer is added. The pipette tip is replaced each time the medium is changed to avoid cross-contamination.

[0027] Furthermore, during the culture process described in step (3), when obvious clones of the primordial germ cells appear, they are passaged at a ratio of 1:4 to 1:2, or transferred to culture plates with larger pore sizes for amplification. When the cell number reaches 1×10⁻⁶, the culture continues. 5 Once the cell line is successfully established, it can be used for further processing.

[0028] This invention discloses a method for establishing a lineage of female primordial germ cells in poultry. The method includes: obtaining fertilized eggs incubated for 5.5–7.5 days; removing the embryos and identifying their sex; isolating the left gonadal tissue from the female embryo; digesting and grinding the tissue to obtain unilaterally derived primordial germ cells; and culturing the left side separately to establish a stable, passaged female primordial germ cell line. Compared with existing methods for isolating primordial germ cells, this invention improves the culture stability and clone-forming ability of female primordial germ cells, facilitating long-term culture and expansion of these cells. It can be applied to fields such as poultry genetic improvement, germplasm resource preservation, and gene-editing breeding.

[0029] This invention utilizes the physiological mechanism of asymmetrical gonadal development in female chicken embryos. Only the left gonad in a female chicken embryo can develop into a functional ovary, with strong proliferative activity and low apoptosis levels of progenitor cells (PGCs) within the gonad; while the right gonad exhibits high apoptosis levels. If samples are taken from both gonads, it will result in difficulty in forming female PGC clones and a high failure rate in establishing a clonal line. Figure 1 and Figure 2 It can be seen that: Figure 1 The female's right gonad failed to establish a lineage; Figure 2The establishment of PGCs derived from the right female gonad was unstable, with a large number of cell debris and apoptotic cells appearing. In other words, the establishment of the right female gonadal line failed, and a large number of cell debris and apoptotic cells were present during the establishment process. Current techniques do not distinguish between the left and right female chicken embryo gonads, ignoring the physiological characteristic of asymmetrical development of the female chicken embryo gonads. The right gonadal germ cells exhibit severe apoptosis, and their introduction into the culture system will destroy the activity of female germ cells, affecting the establishment of the line.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0031] 1. This invention employs separate left-side culture, which significantly improves the culture stability and line establishment success rate of female PGCs compared to existing methods for isolating gonads. The left-side gonad-derived primordial germ cells obtained by this invention exhibit more stable proliferative capacity and more typical clonal morphology.

[0032] 2. The female primordial germ cell line obtained by this invention is beneficial to improving the application value of female PGCs in poultry genetic improvement, germplasm resource preservation and gene editing breeding. Attached Figure Description

[0033] Figure 1 These are morphological diagrams of female primordial germ cells. A shows the morphological diagram of PGCs derived from the left female gonad after cell line establishment; B shows the morphological diagram of PGCs derived from the right female gonad.

[0034] Figure 2 These are morphological diagrams of female primordial germ cells. A shows the morphological diagram of PGCs derived from the left female gonad after cell line establishment; B shows the morphological diagram of PGCs derived from the right female gonad.

[0035] Figure 3 These are morphological diagrams of male primordial germ cells after establishment. A shows the cell morphology of PGCs derived from the left male gonad after establishment; B shows the cell morphology of PGCs derived from the right male gonad after establishment.

[0036] Figure 4 Figure 1 shows the results of apoptosis detection of PGCs from different sexes and different lateral gonads. A is the flow cytometry diagram of apoptosis of PGCs from the left gonad (FL) and right gonad (FR) of the maternal embryo; B is the flow cytometry diagram of apoptosis of PGCs from the left gonad (ML) and right gonad (MR) of the male embryo; C is the flow cytometry diagram of apoptosis of PGCs from the left gonad (ML) of the male embryo and the left gonad (FL) of the maternal embryo; D is the flow cytometry diagram of apoptosis of PGCs from the right gonad (MR) of the male embryo and the right gonad (FR) of the maternal embryo.

[0037] Figure 5This image shows the results of indirect immunofluorescence staining to detect the expression of PGC-specific proteins. The images show the results of immunofluorescence labeling of PGC-specific genes DDX4, EMA-1, SOX2, SSEA-1, and DAPI for labeling cell nuclei. The image labeled "Merge" is a combined image of the two images on the left. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but this should not be construed as limiting the present invention.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0041] The feederless culture medium consists of KO-DMEM basal medium (KnockOut). TM DMEM (ThermoScientific™, catalog number 10829018) is fortified with the following ingredients: 1×B-27, 2 mM GlutaMAX, 1×Non-essential amino acid solution, 0.1 mM β-mercaptoethanol, 1×Nucleoside, 1.2 mM Sodium pyruvate, 0.2% Ovalbumin, 0.2% Sodium heparin, 25 ng / mL Human activin A, 4 ng / mL FGF2, 0.2% Chicken serum, and 1% Penicillin-Streptomycin antibiotics.

[0042] Example 1

[0043] Lateral separation and establishment of primordial germ cells from left and right lateral gonads in chicken embryos

[0044] Fresh fertilized eggs were selected and incubated in an incubator at 37–38℃ and 58% relative humidity for 5.5–7.5 days. After sterilizing the eggshells with 75% alcohol, the eggshells were opened and the chicken embryos were removed. The embryos were placed in a dish containing PBS to wash away any blood. Then, under a stereomicroscope, the left and right gonads of the chicken embryos were carefully dissected and separated, and placed in centrifuge tubes containing 200 μL of PBS (pH 7.0). Simultaneously, a 0.5 × 0.5 mm tissue sample was taken with fine forceps and placed in a PCR tube for PCR sex determination. CHDZ-specific primers (gene number: NM_204941.2) were used as shown in Table 1 below. The PCR amplification reaction system is shown in Table 2 below.

[0045] Table 1: Primer sequences for chicken embryo sex determination

[0046]

[0047] Table 2: PCR reaction system (10 μL)

[0048]

[0049] After mixing, perform the PCR reaction. The PCR reaction procedure is as follows:

[0050] 98°C for 2 min; 98°C for 10 s, 60°C for 15 s, 68°C for 40 s, 40 cycles; store at 4°C.

[0051] After mixing the PCR product with 10×DNA loading buffer (Takara), 5 μL of the mixture was subjected to 1% agarose gel electrophoresis. During electrophoresis, markers were used to compare the size of the bands. Males showed a single band (600 bp), while females showed two bands (600 bp and 450 bp).

[0052] The PBS-containing centrifuge tubes were briefly centrifuged and the PBS discarded. 200 μL of 0.25% trypsin was added for digestion, followed by grinding with a grinding rod. Digestion was then terminated by adding DMEM containing 10% FBS. The cells were centrifuged at 1500 rpm for 5 min, and the cell pellet was collected and resuspended. Left and right gonad-derived cells were seeded into 48-well plates containing 300 μL of feeder-free medium and cultured at 37°C, 5% CO2, and 60–70% relative humidity. During the initial culture period, half of the culture medium was changed every two days. When changing the medium, the 48-well plate was tilted on the workbench, and 150 μL of liquid was gently aspirated from the surface layer of each well using a 200 μL pipette and discarded. The pipette tip was changed before moving to the next well. After aspirating from all wells, 150 μL of preheated (37°C) feeder-free medium was added to each well, and the plate was placed in an incubator at 37°C, 5% CO2, and 60–70% relative humidity for continued culture.

[0053] When an excessive number of adherent cells negatively impacts the growth of primordial germ cells, the cell suspension in the wells is collected, the bottom is digested with Accutase, centrifuged, and resuspended in feeder-free medium for further culture. The growth and colony formation of different groups of PGCs are observed under an inverted microscope. When primordial germ cells show clear colonies, they are passaged at a 1:2 ratio or transferred to larger-well plates for expansion. When the cell number reaches 1×10⁶ cells / well... 5 At this point, the cell line establishment is considered successful. Figure 1 A, Figure 2 A, Figure 3 A and B.

[0054] Observation results of female PGCs as follows Figure 1 , 2. Figure 1 Only PGCs derived from the left gonad were successfully established; those derived from the right gonad failed. Figure 2 PGCs derived from the left gonad were successfully established, while those from the right gonad failed. The right gonad culture background contained numerous clonal clusters but also a large number of cell debris and apoptotic cells. Therefore, the establishment of PGCs derived from the female right gonad is unstable. The results of establishing PGCs from the left and right gonads of the four groups of female chicken embryos are detailed in Table 3.

[0055] Table 3: Statistical analysis of PGC establishment results from left (L) and right (R) gonads of different female chicken embryos

[0056]

[0057] Table 3 shows that multiple attempts to establish PGCs derived from the left gonad were successful, while multiple attempts to establish PGCs derived from the right gonad failed. The results of observation of male PGCs are as follows: Figure 3 PGCs isolated from the left and right gonads of male chicken embryos exhibit excellent clonogenic ability, forming numerous, large, and dense clonal clusters with stable overall cell structure. Compared to female PGCs, PGCs isolated from the left and right gonads of males demonstrate even stronger clonogenic ability, with no significant difference between the two sides.

[0058] In this experiment, the feeder-free culture medium consisted of KO-DMEM basal medium supplemented with 1×B-27, 2mM GlutaMAX, 1×MEM non-essential amino acid solution, 0.1 mM β-mercaptoethanol, 1×nucleoside, 1.2 mM sodium pyruvate, 0.2% ovalbumin, 0.2% heparin sodium, 25 ng / ml human activin A, 4 ng / ml FGF2, 0.2% chicken serum, and 1% penicillin-streptomycin antibiotics.

[0059] Example 2

[0060] Apoptosis level detection of PGCs from different lateral gonadal origins

[0061] Fresh fertilized eggs were selected and incubated in an incubator at 37–38℃ and 58% relative humidity for 5.5–7.5 days. After sterilizing the eggshells with 75% alcohol, the chicken embryos were removed and placed in a dish containing PBS to wash away any blood. Then, under a stereomicroscope, the left and right gonads of the chicken embryos were carefully dissected and separated, and placed in centrifuge tubes containing PBS. The left and right gonads were isolated and sexed by PCR, and the embryos were divided into four groups based on sex and whether they were left or right: left gonad of maternal embryo (FL), right gonad of maternal embryo (FR), left gonad of male embryo (ML), and right gonad of male embryo (MR). The gonadal tissues from each group were digested with trypsin, washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking the cells at room temperature for 1 h, TUNEL reaction solution was added, and the cells were incubated at 37℃ in the dark for 1 h. No TdT enzyme was added to the negative control group. Subsequently, primary antibody DDX4 (Biorbyt, orb5967) was added and incubated overnight at 4°C in the dark. Then, secondary antibody Goat Anti-Rabbit IgG (H+L) FITC-conjugated (Affinity Biosciences LTD, S0008) was added and incubated at room temperature for 1 h. Finally, flow cytometry was used to detect the apoptosis levels in each group. The results showed... Figure 4 : Figure 4 A. In females, the level of apoptosis in the left gonad is lower than that in the right gonad, exhibiting an asymmetrical apoptosis pattern; such as Figure 4 B. There was no significant difference in the level of apoptosis between the left and right gonads in males; Figure 4 C Figure 4 Results D showed that there was no significant difference in apoptosis levels between females and males in the left gonad, while the apoptosis level in females was significantly higher than that in males in the right gonad.

[0062] Example 3

[0063] Identification of female PGC cell lines: indirect immunofluorescence

[0064] In Example 1, after successful establishment of the PGC line, female PGCs were collected, washed three times with PBS, and then fixed on 0.1% poly-L-lysine-coated slides with 4% paraformaldehyde for 30 min until the cells adhered to the slides. The cells were then washed twice with PBS. Next, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 15 min at room temperature and incubated for 1 h at room temperature in blocking buffer (PBS containing 1% BSA). Then, the PGCs were incubated overnight at 4°C in blocking buffer containing antibodies including DDX4 (Biorbyt, orb5967), EMA-1 (Developmental Studies Hybridoma Bank), SOX2 (SantaCruz Biotechnology, sc-398254), and SSEA-1 (Developmental Studies Hybridoma Bank, MC-480). After three PBS washes, the PGCs were incubated with secondary antibodies at room temperature for 1 h. Finally, the PGCs were stained with DAPI and washed three times with PBS. After adding 10 μL of anti-fluorescence quencher, the slide was sealed with nail polish and observed using a fluorescence microscope.

[0065] Combination Figure 5 Indirect immunofluorescence staining results showed that the successfully isolated and cultured cells were PGCs. These cells specifically expressed germline markers (DDX4 / EMA-1) and pluripotency markers (SOX2 / SSEA-1) of PGCs, proving that the cells isolated and cultured by the method of this invention are female PGCs.

Claims

1. A method for establishing a lineage of female primordial germ cells in poultry, characterized in that, Includes the following steps: (1) Sampling; fertilized eggs are placed in an incubator and incubated for 5.5 to 7.5 days. The embryos of the fertilized eggs are removed and the left gonad is separated under a stereomicroscope. A small amount of tissue is taken for sex identification. (2) Cell separation; digest and grind the left gonad, and stop digestion with complete culture medium after digestion; collect the bottom cells by centrifugation, and resuspend to obtain a suspension of primordial germ cells from the left gonad; (3) Cell culture: The cells derived from the left gonad were seeded in a culture medium without a feeder layer and cultured continuously to complete the establishment of the cell line.

2. The method for establishing poultry female primordial germ cell lines according to claim 1, characterized in that, In step (1), when the eggshell is incubated for 5.5 to 7.5 days, the eggshell surface is disinfected with alcohol, the eggshell is opened and the chicken embryo is taken out. The embryo is placed in a dish containing PBS, the blood on the embryo is washed off, the left gonad is found under a stereomicroscope, it is separated, and a 0.5×0.5 mm tissue is taken with fine tweezers and placed in a PCR tube.

3. The method for establishing poultry female primordial germ cell lines according to claim 1, characterized in that, In step (1), the sex determination method is preferably performed by direct PCR amplification.

4. The method for establishing poultry female primordial germ cell lines according to claim 1, characterized in that, In step (2), the complete culture medium is DMEM medium containing 10% FBS.

5. The method for establishing poultry female primordial germ cell lines according to claim 1, characterized in that, In step (3), the feederless culture medium consists of KO DMEM basal medium (KnockOut). TM DMEM, Thermo Scientific TM The following ingredients were added: B-27, GlutaMAX, MEM non-essential amino acid solution, β-mercaptoethanol, nucleoside, sodium pyruvate, ovalbumin, sodium heparin, Human activin A, FGF2, chicken serum and bispecific antibodies.

6. The method for establishing poultry female primordial germ cell lines according to claim 1, characterized in that, The continuous culture mentioned in step (3) refers to continuous culture under the conditions of 37℃, 5% CO2, and 60~70% relative humidity.

7. The method for establishing a lineage of female primordial germ cells in poultry according to claim 1, characterized in that, In step (3), half of the culture medium is replaced every two days during the continuous culture process. When replacing the medium, the well plate containing the culture medium is tilted, the old culture medium is taken out from the top layer of the liquid, and then preheated fresh culture medium without feeder layer is added. The pipette tip is replaced each time the medium is changed to avoid cross-contamination. When too many adherent cells affect the growth of primordial germ cells, the adherent somatic cells at the bottom of the culture plate are digested with Accutase enzyme to passage the primordial germ cells.

8. The method for establishing a lineage of female primordial germ cells in poultry according to claim 1, characterized in that, During the continuous culture process described in step (3), when obvious clones of the primordial germ cells appear, they are passaged or transferred to culture plates with larger pore sizes for expansion. When the cell number reaches 1×10⁻⁶, the culture continues. 5 The cell line was determined to be successfully established at this time.

Images