Method for improving transfection efficiency of chicken primordial germ cells

Abstract

The invention discloses a method for improving transfection efficiency of chicken primordial germ cells, and belongs to the technical field of cell transfection. The invention provides application of an EGFR (epidermal growth factor receptor) activator NSC 228155 in improvement of plasmid uptake efficiency and cationic liposome transfection efficiency of chicken PGCs, and a method for efficiently transfecting the chicken PGCs is constructed. Experimental results show that compared with a control group, the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a DMRIE-C transfection reagent and the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a LipofectamineTM 3000 transfection reagent are both remarkably improved, which indicates that the EGFR activator remarkably improves the transfection efficiency of the chicken PGCs; and the EGFR activator does not influence the cell characteristics of the chicken primordial germ cells. The method lays a foundation for establishing an efficient gene editing chicken technical system, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of cell transfection, and in particular to a method for improving the transfection efficiency of chicken primordial germ cells. Background Art

[0002] In recent years, chicken germplasm resources have undergone genetic evolution, resulting in changes in behavioral traits, trait adaptability, reproductive characteristics, and morphological structure, making local chicken breeds of particular conservation value. Chicken primordial germ cells (PGCs) are precursor cells capable of developing into both female and male germ cells. They possess the ability to preserve genetic material and stably transmit this information to offspring. Recently, there have been studies exploring cryopreservation of PGCs to preserve germplasm resources, as well as gene editing of PGCs to generate gene-edited chickens. The transfection method is crucial for the success of gene editing. Cationic liposomes are one of the most common vectors used in chemical transfection. Cationic liposomes not only effectively protect genetic material from nuclease degradation but also promote endosomal escape. Due to their ease of synthesis and formulation, as well as their simple transfection procedures, cationic liposomes have become highly attractive gene delivery vehicles. Although cationic liposomes are currently the most efficient non-viral gene delivery vehicles, their transfection efficiency remains significantly lower than that of viral vectors, hindering the application of gene-edited chicken technology. Therefore, there is an urgent need to develop methods to improve the transfection efficiency of PGCs via cationic liposome transfection.

[0003] EGFR (epidermal growth factor receptor) belongs to the ErbB family of tyrosine kinase receptors and is a membrane transport glycoprotein. EGFR is believed to be crucial for cell proliferation, cell migration, cell cycle, and transmembrane transport of exogenous substances. EGFR is usually located on the surface of the cell membrane. When bound to a ligand (such as epidermal growth factor), it undergoes dimerization and phosphorylation, activating downstream signaling pathways. The small molecule compound NSC 228155 is an activator of EGFR with a CAS accession number of 113104-25-9. It generates H2O2 through dimerization of SOD1, which interacts with cell surface cysteines, promoting EGFR phosphorylation, thereby activating EGFR and then activating downstream pathways through the EGFR signaling pathway.

[0004] Currently, there are no reports on the regulation of PGCs by EGFR activators. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the transfection efficiency of chicken primordial germ cells to solve the problems existing in the above-mentioned prior art. The EGFR activator provided by the present invention significantly improves the cationic liposome transfection efficiency and plasmid uptake efficiency of chicken PGCs.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of an epidermal growth factor receptor activator in improving the plasmid uptake ability of chicken primordial germ cells.

[0008] Preferably, the epidermal growth factor receptor activator is NSC 228155.

[0009] The present invention also provides an application of an epidermal growth factor receptor activator in improving the transfection efficiency of chicken primordial germ cells.

[0010] Preferably, the epidermal growth factor receptor activator is NSC 228155.

[0011] Preferably, the transfection reagent is cationic liposome.

[0012] Preferably, the cationic liposome is DMRIE-C or Lipofectamine TM 3000.

[0013] The present invention also provides a method for improving the transfection efficiency of chicken primordial germ cells, comprising the step of treating the chicken primordial germ cells with an epidermal growth factor receptor activator.

[0014] Preferably, the epidermal growth factor receptor activator is NSC 228155; the treatment concentration of NSC 228155 is 1 μM;

[0015] And / or, the treatment time is 40 minutes.

[0016] The present invention also provides a method for transfecting chicken primordial germ cells, comprising the following steps:

[0017] treating the chicken primordial germ cells with an epidermal growth factor receptor activator to obtain treated cells;

[0018] The treated cells were transfected using cationic liposomes.

[0019] Preferably, the epidermal growth factor receptor activator is NSC 228155; the treatment concentration of NSC 228155 is 1 μM;

[0020] And / or, the treatment time is 40 minutes.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides the application of EGFR activator NSC 228155 in improving the plasmid uptake efficiency and cationic liposome transfection efficiency of chicken PGCs, and constructs a method for efficiently transfecting chicken PGCs. The experimental results show that the gene transfection efficiency of chicken PGCs treated with EGFR activator NSC 228155 increased from 4.7% to 10.7% under DMRIE-C transfection reagent compared with the control group, and increased from 4.7% to 10.7% under Lipofectamine 20. TM The gene transfection efficiency using 3000 transfection reagent increased from 7.04% to 18.8%, demonstrating that the EGFR activator significantly enhances the transfection efficiency of chicken PGCs. The method provided by this invention improves the transfection efficiency of chicken PGCs without affecting the cellular properties of the PGCs themselves. This invention lays the foundation for establishing an efficient gene-editing technology system for chickens and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The effect of EGFR activators on the toxicity of PGCs; NSC is NSC 228155; ns represents no statistically significant difference, * represents P < 0.05, which indicates a statistically significant difference; ** represents P < 0.01, which indicates a statistically extremely significant difference;

[0025] Figure 2 Fluorescence microscopic observation of the effect of different concentrations of EGFR activators on the transfection efficiency of PGCs; NSC refers to NSC228155; Non-treated refers to treatment without activator; Bright refers to bright field, EGFP refers to enhanced green fluorescent protein; Merged refers to merged field; scale length is 100 μm;

[0026] Figure 3 The figure shows the flow cytometry analysis results of the effect of different concentrations of EGFR activators on the transfection efficiency of PGCs; NSC refers to NSC 228155; NT refers to no activator treatment; EGFP Proportion refers to the ratio of enhanced green fluorescent protein;

[0027] Figure 4Statistical results of the effects of different concentrations of EGFR activators on PGCs transfection efficiency; NSCConcentration is the concentration of NSC 228155; NT is no activator treatment; EGFP is enhanced green fluorescent protein; *P<0.05, ***P<0.001;

[0028] Figure 5 The figure shows the flow cytometry analysis results of the effect of EGFR activators on PGCs transfection efficiency at different treatment times; NSC refers to NSC 228155; NT refers to no activator treatment; EGFP Proportion refers to the ratio of enhanced green fluorescent protein;

[0029] Figure 6 The statistical results of the effect of EGFR activators on PGCs transfection efficiency at different treatment times are shown in the figure; Treatment time with NSC is the treatment time of NSC 228155; EGFP is enhanced green fluorescent protein; *P<0.05, **P<0.01, ***P<0.001, ns is P>0.05;

[0030] Figure 7 Fluorescence microscopic observation of the effect of EGFR activators on the efficiency of PGCs transfected with large plasmids; NSC refers to NSC228155; NT refers to no activator treatment; Bright refers to bright field, EGFP refers to enhanced green fluorescent protein; Merged refers to merged field; scale bar length is 100 μm;

[0031] Figure 8 The figure shows the flow cytometry analysis results of the effect of EGFR activators on the efficiency of PGCs transfected with large plasmids; NSC is NSC228155; NT is no activator treatment; EGFP Proportion is the ratio of enhanced green fluorescent protein;

[0032] Figure 9 The statistical results of the effect of EGFR activators on the efficiency of PGCs transfected with large plasmids are shown in the figure; NSC is NSC228155; NT is no activator treatment; EGFP is enhanced green fluorescent protein; ***P<0.001;

[0033] Figure 10 Fluorescence microscopic observation of the effect of EGFR activator on the efficiency of Lipo3000 transfection of PGCs; NSC is NSC 228155; NT is no activator treatment; Bright is bright field, EGFP is enhanced green fluorescent protein; Merged is merged field; scale length 100 μm;

[0034] Figure 11The figure shows the flow cytometry analysis results of the effect of EGFR activators on the efficiency of Lipo3000 transfection of PGCs; NSC refers to NSC 228155; NT refers to no activator treatment; EGFP Proportion refers to the ratio of enhanced green fluorescent protein;

[0035] Figure 12 The statistical results of the effect of EGFR activators on the efficiency of Lipo3000 transfection of PGCs; NSC refers to NSC 228155; NT refers to no activator treatment; EGFP refers to enhanced green fluorescent protein; ***P<0.001;

[0036] Figure 13 Fluorescence microscopic observation of the effect of EGFR activators on the ability of cells to take up plasmids; NSC refers to NSC228155; NT refers to treatment without activator; Cy3-DNA refers to Cy3 fluorescently labeled plasmid; Nucleus refers to nuclear staining; Merged refers to a mixed field of view; scale bar length is 100 μm;

[0037] Figure 14 The statistical results of the effect of EGFR activators on the ability of cells to uptake plasmids are shown in the figure; NSC refers to NSC228155; NT refers to no activator treatment; Percent of plasmid uptake refers to the percentage of plasmid uptake; ***P<0.001;

[0038] Figure 15 This is the result of agarose gel electrophoresis of functional genes showing the effect of EGFR activators on PGCs characteristics;

[0039] Figure 16 The immunofluorescence staining results show the effect of EGFR activators on the characteristics of PGCs; the scale bar is 20 μm;

[0040] Figure 17 This figure shows the effect of EGFR activators on the migration ability of PGCs. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] In order to study the effect of EGFR activators on the transfection efficiency of PGCs, the present invention uses EGFR activator NSC 228155 as an activator of the EGFR signaling pathway. First, the concentration of the EGFR activator for PGCs was verified by MTT cytotoxicity experiment, and it was found that at concentrations of 0.5μM and 1μM, the EGFR activator had no significant toxicity to PGCs. Then, based on the results of the toxicity experiment, PGCs were treated with EGFR activators at different concentrations and times and then transfected. The optimal concentration of the EGFR activator for PGCs was determined, and the transfection efficiency of PGCs was successfully increased from 4.70% to 11.10%, providing a new idea for improving the transfection efficiency of PGCs.

[0047] The present invention has found that the use of Lipofectamine, which is also a cationic liposome TM The 3000 reagent combined with an EGFR activator to treat PGCs further increased the transfection efficiency to 18.8±3%. This study demonstrates that the use of an EGFR activator significantly improves the transfection efficiency of PGCs using transfection reagents of different cationic liposome types.

[0048] In order to explore whether the method of treating cells with EGFR activators can also improve the transfection efficiency of large plasmids, the present invention treated PGCs with NSC and then transfected them with large plasmids (13671bp). The results showed that activating the EGFR signaling pathway also had an improving effect on the transfection of PGCs with large plasmids.

[0049] The present invention also designed an experiment on cell uptake of plasmids. By transfecting Cy3 fluorescently labeled plasmids, it was found that after treatment with EGFR activators, the ability of PGCs to take up plasmids was significantly improved. This result shows that EGFR activators improve transfection efficiency by enhancing the ability of cells to take up plasmids.

[0050] The present invention also explores the cellular characteristics of chicken primordial germ cells treated with an EGFR activator, and verifies the expression of germ cell-related genes of PGCs by RT-PCR, showing that the internal reference gene β-actin is stably expressed in the three groups of CEF, WT PGC, and PGC+NSC. At the same time, the expression of germ cell-related genes CDH, CVH and DAZL and pluripotency genes NANOG and PouV are detected in the two groups of WT PGC and PGC+NSC. At the same time, the group of cells is subjected to immunofluorescence staining, and the group of cells is subjected to immunofluorescence staining for germ stem cell marker gene proteins. The results show that DAZL, SSEA1 and CVH are all positive, indicating that the EGFR signaling pathway is activated, and the germ cell marker genes and cellular characteristics of PGCs are unchanged at the molecular and protein levels.

[0051] The present invention studies the effect of EGFR activators on the migration ability of chicken primordial germ cells. GFP-PGCs expressing green fluorescent protein (GFP) were used as tracer cells to observe the location of PGCs within chicken embryos. GFP-PGCs were treated with an EGFR activator and then transfected with a plasmid, followed by 48 hours of culture. The GFP-PGCs were then injected into the hearts of recipient chicken embryos at the HH15 stage. The embryos were returned to an incubator and incubated until the HH31 stage. The embryos were then dissected and their gonads isolated. A large number of GFP-PGCs were observed colonizing the gonads of the recipient chicken embryos. The experimental results demonstrate that even after treatment with the EGFR activator, PGCs retain their ability to migrate and colonize the gonads.

[0052] 1. Experimental Materials

[0053] 1. Biomaterials

[0054] The mKO PGCs used in the present invention were derived from Guangxi yellow-feather chickens and have been published in Wang, L et al. "Derivation and characterization of primordial germ cells from Guangxi yellow-feather chickens. Poultry science vol. 96, 5 (2017): 1419-1425." The GFP-PGCs used have been published in Xie, Long et al. "An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs." Journal of biological engineering vol. 18, 15.11 Jan. 2024.

[0055] The mouse embryonic fibroblasts (MEFs) used in this invention are derived from 13.5-day-old CD-1 pregnant mice and are described in Xie, Long et al., "Derivation of chicken primordial germ cells using an indirect co-culture system." Theriogenology, vol. 123 (2019): 83-89. The applicants commit to distributing these biological materials to the public within 20 years from the filing date of this application.

[0056] 2. Experimental Reagents

[0057] The reagents used in the present invention are shown in Table 1.

[0058] Table 1 Experimental reagent information

[0059]

[0060] The components of the mKO culture medium of the present invention are shown in Table 2:

[0061] Table 2 mKO culture medium composition

[0062]

[0063] The components of the cKO culture medium of the present invention are shown in Table 3:

[0064] Table 3 cKO culture medium composition

[0065]

[0066]

[0067] This invention uses the small molecule compound NSC 228155 as an EGFR activator. Unless otherwise specified, the EGFR activator and NSC 228155 in the examples of this invention have the same meaning. To prepare the NSC 228155 stock solution: Centrifuge a centrifuge tube containing 1 mg of NSC 228155 powder at 5000 rpm for 10 minutes, allowing all the powder on the tube top and walls to settle to the bottom. Add 344.5 μL of DMSO to the centrifuge tube and pipette until the powder is completely dissolved. This creates a 10 mM NSC 228155 stock solution. Aliquot and store at -80°C.

[0068] The reagents used for RT-PCR, immunofluorescence staining, and cell injection of the present invention are shown in Table 4:

[0069] Table 4 Reagents used for RT-PCR, immunofluorescence staining, and cell injection

[0070]

[0071] 3. Experimental instruments

[0072] The experimental instrument information of the present invention is shown in Table 5:

[0073] Table 5 Experimental instrument information

[0074]

[0075]

[0076] 2. Experimental Methods of the Present Invention

[0077] 1. Cell culture method of the present invention

[0078] 1.1 MEF culture and treatment

[0079] MEF cells are used as a feeder layer in the present invention to provide nutrition for PGCs cells. After thawing, MEFs are placed in a 100mm culture dish for culture. When the cells are full, they are passaged at a ratio of 1:3. When the MEFs are passaged to the P3 generation and the confluence reaches 80-90%, mitomycin C (MMC) is added according to the amount of MEF cell culture medium so that the MMC reaches a final working concentration of 10μg / mL. After treating the cells with MMC for 2 hours, the culture medium is washed away, washed 3 times with PBS, and transferred to a 6-well plate at a ratio of 1:6. It can be used after at least 2 days. The treated MEF needs to change the medium the next day, and the medium should be changed every two days if it is not used.

[0080] 1.2 Culture of mKO PGC cells

[0081] PGCs are suspension cells. mKO PGC culture utilizes a three-dimensional culture method. MEFs treated with mitomycin C are laid on the bottom of the culture dish, and PGCs are separated from MEFs using an insert. When changing the cell culture medium, 1 mL of culture medium outside the insert is aspirated and discarded, and 1 mL of fresh mKO culture medium is added to the outside of the insert. The typical passage time is 3 days. During passage, use sterilized forceps to move the insert to a well containing MEFs treated with MMC 2 days earlier. Transfer 1 mL of culture medium from the original well to a new well, and then add 1.5 mL of fresh culture medium to the new well.

[0082] 2. Transfection method of cationic liposome DMRIE-C of the present invention:

[0083] Dilute 0.1 μL of the above EGFR activator stock solution with 1 mL of mKO culture medium and mix thoroughly to form a premix. Collect 1×10 5 PGCs cells were placed in a centrifuge tube, centrifuged for 3 minutes, resuspended with the premixed solution, incubated for 40 minutes, then centrifuged at 1200 rpm for 3 minutes, and washed twice with PBS.

[0084] a. Prepare two 1.5 mL centrifuge tubes and label them A and B.

[0085] b. Add 250 μL of Opti-MEM culture medium to tubes A and B respectively, add 6 μL of DMRIE-C to tube A, and mix thoroughly.

[0086] c. Add 5 μg of plasmid to tube B and mix thoroughly.

[0087] d. Transfer the liquid in tube B to tube A, mix gently, and incubate at room temperature for 45 minutes.

[0088] e. During the incubation process, 1×10 6 PGCs cells were placed in a centrifuge tube, centrifuged at 1200 rpm for 3 min, washed once with PBS, resuspended in 100 μL cKO culture medium, and transferred to a 24-well plate.

[0089] f. After the incubation is completed, add the incubated liquid to the cell culture medium.

[0090] After 7 hours, collect the cells into a centrifuge tube, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, resuspend in 1 mL of fresh mKO culture medium, and place in a 24-well plate pre-plated with MMC-treated MEFs. Observe after 48 hours.

[0091] 3. Cationic liposome Lipofetamine of the present inventionTM 3000 transfection method:

[0092] a. Collect 1×10 5 PGCs cells were placed in a centrifuge tube, centrifuged at 1200 rpm for 3 min, and resuspended in 100 μL cKO culture medium.

[0093] b. Add 5 μg of plasmid to an EP tube containing 125 μL of Opti-MEM culture medium, and add 10 μL of P3000 reagent. Mix gently and mark the tube as A.

[0094] c. Add 3.75 μL of Lipofectamine TM 3000 μL was added to an EP tube containing 125 μL Opti-MEM culture medium, mixed gently, and marked as tube B.

[0095] d. Transfer the liquid in tube A to tube B, mix gently, and incubate for 15 minutes.

[0096] e. After the incubation is completed, add the incubated liquid to the cell culture medium.

[0097] After 7 hours, collect the cells into a centrifuge tube, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, resuspend in 1 mL of fresh mKO culture medium, and place in a 24-well plate pre-plated with MMC-treated MEFs. Observe after 48 hours.

[0098] 4. Flow analysis method of the present invention:

[0099] To collect cells, pipette the cells directly from the well into a centrifuge tube. Rinse the culture dish with 1 mL of PBS and transfer to the same centrifuge tube, collecting as many cells as possible. Centrifuge at 1200 rpm for 3 minutes, resuspend in 1 mL of PBS, pass through a 35 μm sieve, and transfer to a flow cytometer. Load the sample onto the flow cytometer, turning on the laser channel corresponding to the GFP fluorescent protein before loading.

[0100] 5. RNA extraction, reverse transcription and RT-PCR analysis methods of the present invention

[0101] Cellular RNA extraction: Strictly follow the instructions for the RNAeasy™ Animal RNA Extraction Kit. Briefly, the steps are as follows: Collect cells into a centrifuge tube and centrifuge at 1200 rpm for 3 minutes. Discard the supernatant and wash twice with PBS. Add 300 μL of lysis buffer and pipette until completely clear. Then, add an equal volume of binding buffer and gently invert to mix. Transfer the liquid to a purification column and centrifuge at 12,000 rpm for 3 minutes. Discard the liquid in the collection tube. Add 600 μL of Wash Buffer I and centrifuge at 12,000 rpm for 3 minutes. Discard the liquid in the collection tube. Add 600 μL of Wash Buffer II and centrifuge at 12,000 rpm for 3 minutes. Discard the liquid in the collection tube. Repeat the previous step. Centrifuge at 14,000 rpm for 2 minutes to remove any residual liquid. Elute with 30 μL of Elution Buffer to obtain purified RNA.

[0102] RNA reverse transcription: After measuring the RNA concentration using a microplate reader, prepare the reverse transcription reaction system based on a total RNA volume of 500 ng. The configuration table is as follows:

[0103] Table 6 Reverse transcription reaction system

[0104]

[0105] The reverse transcription system was performed according to the instructions of the TranScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Full Gold / S20226-V3), and the obtained cDNA was stored at -20°C for a long time. The PCR reaction system was configured according to the following table:

[0106] Table 7 PCR reaction system

[0107]

[0108] Since the Tm values ​​of the PCR primers in the embodiments of the present invention are all around 60°C and the product lengths are all 100-250 bp, the PCR reaction is performed according to the following reaction procedure:

[0109] Table 8 PCR reaction program

[0110]

[0111] Table 9 mKO PGC RT-PCR primers

[0112]

[0113]

[0114] 5. Cell Immunofluorescence Staining Method of the Present Invention

[0115] PGCs were collected, centrifuged, and resuspended in 4% paraformaldehyde for 15 minutes. The fixed PGCs were then evenly smeared onto the hydrophobic barrier surface of a pathology-grade microscope slide, and the slide was circled and bordered with a hydrophobic immunohistochemistry pen. The smeared PGCs were then incubated with a blocking solution consisting of 6% horse serum in PBS for 45 minutes and then incubated with the primary antibody for 60 minutes. After three rinses with PBS, the PGCs were incubated with a secondary antibody for 60 minutes. The slides were then rinsed three times with PBS, and the nuclei were counterstained with ProlongGold antifade reagent containing DAPI. Finally, the treated slides were examined and imaged under a fluorescence microscope. The primary antibodies used were mouse anti-SSEA1, rabbit anti-DAZL polyclonal antibodies (prepared in our laboratory), and rabbit anti-CVH polyclonal antibodies (prepared in our laboratory), diluted 1:200 in blocking solution. Secondary antibodies used were goat anti-mouse IgG conjugated with Alexa Fluor 594 and goat anti-rabbit IgG conjugated with DyLight 488, respectively.

[0116] 6. The method of cell chicken embryo injection and cell migration of the present invention

[0117] First, prepare the cells. Use the GFP-PGCs cells with green fluorescent protein cultured in this laboratory to study the migration ability of PGCs. Treat GFP-PGCs with EGFR activator for 40 minutes and then transfect the plasmid. Then continue to culture for 48 hours before injection. GFP-PGCs cells without any treatment serve as a control. Next, prepare the chicken embryos for injection. First, place the eggs in a 16°C refrigerator for 12-16 hours to synchronize their embryonic stages. Then, incubate the eggs in an incubator at 38°C and 60% humidity for 55 hours. According to the cell density of 1×10 6 cells / mL to collect cells. Then add trypan blue to the cell suspension at a ratio of 1:10 and mix well. Make cell drops of 100 μL per drop of the cell suspension in a 100mm culture dish, and add 10 mL of mineral oil to the culture dish to cover the cell drops. Open a window with a diameter of about 1 cm in the center of the air chamber of the 55h incubated egg, drip 200 μL of PBS containing 1% double antibody, and then carefully lift the inner eggshell membrane of the egg with sterile tweezers, and use a glass needle with a diameter of 20 μm to aspirate about 1×10 4 The cells were injected into the heart of the chick embryo. The window in the center of the egg's air chamber was sealed with two layers of parafilm and returned to the incubator for another five days. On the seventh day of incubation, the chick embryo was dissected, the gonads isolated, and observed and photographed under a fluorescence microscope.

[0118] Example 1 MTT cytotoxicity assay

[0119] 1. Experimental Methods

[0120] The cytotoxicity experiment of EGFR activators on PGCs was set up into five groups with concentrations of 0μM, 0.5μM, 1μM, 2μM and 4μM respectively.

[0121] Cytotoxicity was detected using the MTT cytotoxicity detection kit, and the detection steps were carried out according to the instructions:

[0122] First, dissolve 25mg of MTT in 5mL of MTT solvent to prepare a 5mg / mL MTT solution for later use. Prepare PGCs cells and place them in a 96-well plate, with 5000 cells per well and 3 wells per group. Add EGFR activators according to the set concentration gradient and continue to culture for 2 hours. During the test, add 10μL of MTT solution to each well and continue incubating in the cell culture incubator for 4 hours. Then, add 100μL of formazan solution to each well, mix appropriately, and continue incubating in the cell culture incubator until the formazan is completely dissolved under an ordinary optical microscope. Measure the absorbance (OD value) at 570nm.

[0123] 2. Experimental Results

[0124] The results are as follows Figure 1 As shown in the figure, under the treatment conditions of 0.5μM and 1μM concentrations of EGFR activator, the activity of cells was not significantly different from that of the control group (P>0.05), while under the treatment conditions of 2μM and 4μM concentrations of EGFR activator, the activity of cells was significantly lower than that of the control group (P<0.05).

[0125] Example 2 Optimization of EGFR activator addition concentration and time

[0126] According to the cytotoxicity results, in order to test the effect of EGFR activators on the gene transfection efficiency of PGCs, cells were treated with 0.5μM and 1μM concentrations of EGFR activators for 40 minutes and then transfected with plasmids; after 48 hours, the cells were observed and photographed under a microscope. The fluorescence microscopy results are shown in Figure 2. Figure 2 As shown; and the transfection efficiency was counted by flow cytometry, and the flow analysis results were shown Figure 3 shown.

[0127] The results are as follows Figure 4 As shown, compared with the control group (4.7%), the EGFR activator at a concentration of 0.5 μM and 1 μM increased the gene transfection efficiency of PGCs to 8.93% and 10.7%, respectively, indicating that the EGFR activator had the best effect at a concentration of 1 μM.

[0128] In order to optimize the action time of EGFR activator on cells, PGCs cells were treated with 1 μM EGFR activator for 30 min, 40 min and 50 min respectively and then transfected with plasmids. The transfection efficiency was calculated by flow cytometry. The results of flow cytometry analysis are shown in Figure 2. Figure 5 The statistical results are shown in Figure 6 As shown in the figure, the transfection efficiency increased from 5.82% in the control group to 9.57%, 11.1% and 8.38% respectively, indicating that the EGFR activator has the best effect on improving the transfection efficiency of PGCs when the treatment time is 40 minutes.

[0129] Subsequent experiments were performed according to the protocol with an EGFR activator concentration of 1 μM and a treatment time of 40 min.

[0130] Example 3 Effect of EGFR Activators on Transfection Efficiency of Plasmids of Different Sizes

[0131] In order to determine the effect of EGFR activator on the efficiency of PGCs transfection with larger fragment plasmids, PGCs were treated with activator and then transfected with PB-EGFP-pMDA5-RIG plasmid (13671 bp); 48 hours later, the cells were observed and photographed under a microscope. The fluorescence microscopy results are shown in Figure 2. Figure 7 As shown; the transfection efficiency was counted by flow cytometry, and the flow cytometry analysis results were shown as follows: Figure 8 shown.

[0132] The statistical results are as follows Figure 9 As shown, the transfection efficiency of the activator group (5.53%) was significantly higher than that of the control group (1.78%), indicating that the EGFR activator also had a significant effect on the transfection of PGCs with a plasmid of 13671 bp (P<0.001).

[0133] Example 4 Effect of EGFR Activators on the Transfection Efficiency of Other Liposome Transfection Reagents

[0134] To determine the effect of EGFR activators on the cationic liposome transfection reagent lipofetamine TM 3000 (abbreviated as Lipo3000) transfection of PGCs has an improvement effect. After treating the cells with EGFR activator, PGCs were transfected with Lipo3000. After 48 hours, the cells were observed and photographed under a microscope. The fluorescence microscopy results are as follows Figure 10 As shown; the transfection efficiency was counted by flow cytometry, and the flow cytometry analysis results were shown as follows: Figure 11 shown.

[0135] The statistical results are as follows Figure 12As shown in the figure, compared with the control group (7.04%), the gene transfection efficiency of PGCs treated with activators increased to 18.8%, indicating that activating the EGFR signaling pathway can significantly improve the transfection efficiency of cationic liposome Lipo3000 transfected PGCs (P<0.001).

[0136] Example 5 Effect of EGFR Activators on Cellular Plasmid Uptake

[0137] 1. Preparation of Cy3 fluorescently labeled plasmid:

[0138] a. Prepare the reaction solution according to the manufacturer's instructions: 37.5 μL of sterile water, 5 μL of 10× Labeling Buffer A, 5 μL of 1 mg / mL plasmid, and 2.5 μL of Cy3 labeling reagent.

[0139] b. Incubate the reaction solution in a 37°C water bath for 1 hour. After incubation for 30 minutes, briefly centrifuge to minimize loss of the reaction solution.

[0140] c. After incubation, add sterile water to the reaction solution to 100 μL. Then, add 0.1 volume of 5 M sodium chloride solution and 2 volumes of pre-chilled anhydrous ethanol. Mix thoroughly and place in a -20°C refrigerator for 30 minutes.

[0141] d. Place the reaction solution in a refrigerated centrifuge at 4°C and centrifuge at 14,000 rpm for 30 min.

[0142] e. Discard the supernatant, wash with 500 μL of room temperature 70% ethanol, and centrifuge again for 30 minutes as in the previous step.

[0143] f. Discard the supernatant and resuspend the suspension in 5 μL of sterile water. Use immediately or store in a -20°C refrigerator for long-term storage.

[0144] 2. Transfection of Cy3 fluorescently labeled plasmid

[0145] Transfection was performed according to the above method, but light was not an issue during the transfection process. Seven hours after transfection, the cells were centrifuged at 1200 rpm for 3 minutes, washed twice with PBS, resuspended in 0.05% Tween-20, incubated for 3 minutes, and centrifuged at 1200 rpm for 3 minutes. The cells were washed twice again with PBS, resuspended in 1 mL of PBS, and 1 μL of Hoechst 33342 live cell stain was added. After staining for 15 minutes, the cells were washed once again with PBS, resuspended in 500 μL of PBS, and placed in a 24-well plate. The cells were allowed to stand for 5 minutes and observed and photographed using an inverted fluorescence microscope. Fluorescence signal intensity was semi-quantitatively analyzed using Image J.

[0146] 3. Experimental Methods

[0147] In order to explore the effect of EGFR activator on the plasmid uptake ability of PGCs, the present invention treated PGCs with EGFR activator and then transfected with Cy3 fluorescent-labeled plasmid. After 7 hours, photos were taken, observed and counted.

[0148] Fluorescence microscopy results Figure 13 As shown, the statistical results are Figure 14 As shown in the results, after treatment with EGFR activators, the proportion of cells with fluorescently labeled plasmids (red fluorescent signals) around the nucleus (51.36%) was significantly higher than that in the control group (26.10%), indicating that activating the EGFR signaling pathway can significantly improve the efficiency of PGCs in taking up plasmids (P<0.001).

[0149] Example 6 Effects of EGFR Activators on PGCs Germ Cell Characteristics

[0150] To further verify the effect of EGFR activators on the germ cell characteristics of PGCs, PGCs cells were first treated with EGFR activators and then transfected with plasmids. The cells were cultured for 48 hours, and then the cells were collected and RNA was extracted. After reverse transcription, RT-PCR was performed to verify the expression of germ cell-related genes in PGCs.

[0151] The results showed that the internal reference gene β-actin was stably expressed in the three groups of CEF, WT PGC, and PGC+NSC, verifying the reliability of the experiment. At the same time, the expression of germ cell-related genes CDH, CVH and DAZL and pluripotency genes NANOG and PouV were detected in the WT PGC and PGC+NSC groups. Figure 15 At the same time, immunofluorescence staining was performed on the cells, and the staining results showed that DAZL, SSEA1 and CVH were all positive. The fluorescence microscopy results were as follows Figure 16 This indicates that activation of the EGFR signaling pathway does not change the germ cell marker genes of PGCs at the molecular and protein levels.

[0152] Example 7 Effect of EGFR Activators on the Migration Ability of Chicken Primordial Germ Cells

[0153] One of the key reasons chicken PGCs can be used as a tool for preparing gene-edited chickens is their ability to migrate to the gonads. Therefore, PGCs must maintain this migratory ability for subsequent applications. To investigate the effects of activating the EGFR signaling pathway on the migratory properties of PGCs, GFP-PGCs expressing green fluorescent protein (GFP) were used as tracer cells to observe the location of PGCs within the chick embryo. GFP-PGCs were first treated with an EGFR activator and then transfected with a plasmid. Cultures were then continued for 48 hours. GPF-PGCs were then injected into the hearts of recipient chick embryos at stage HH15. The embryos were returned to the incubator and incubated until stage HH31. The embryos were then dissected and the gonads isolated.

[0154] The results showed that a large number of GFP-PGCs colonized the gonads of recipient chicken embryos. The fluorescence microscopy results were as follows: Figure 17 These experimental results indicate that after treatment with EGFR activators, PGCs still maintain the ability to migrate and colonize the gonads.

[0155] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of an epidermal growth factor receptor (EGFR) activator in enhancing the plasmid uptake capacity of chicken primordial germ cells.

2. The use according to claim 1, characterized in that The epidermal growth factor receptor activator is NSC228155.

3. Application of an epidermal growth factor receptor activator in improving the transfection efficiency of chicken primordial germ cells.

4. The use according to claim 3, characterized in that The epidermal growth factor receptor activator is NSC228155.

5. The use according to claim 3, characterized in that The transfection reagent is cationic liposome.

6. The use according to claim 5, characterized in that The cationic liposome is DMRIE-C or Lipofectamine TM 3000.

7. A method for improving the transfection efficiency of chicken primordial germ cells, characterized in that: The method comprises the step of treating the chicken primordial germ cells with an epidermal growth factor receptor activator.

8. The method according to claim 7, wherein The epidermal growth factor receptor activator is NSC228155; the treatment concentration of NSC 228155 is 1 μM; And / or, the treatment time is 40 minutes.

9. A method for transfecting chicken primordial germ cells, characterized in that: The following steps are involved: treating the chicken primordial germ cells with an epidermal growth factor receptor activator to obtain treated cells; The treated cells were transfected using cationic liposomes.

10. The method according to claim 9, wherein The epidermal growth factor receptor activator is NSC228155; the treatment concentration of NSC 228155 is 1 μM; And / or, the treatment time is 40 minutes.

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