A method for transfecting chicken primordial germ cells

Abstract

The application discloses a kind of chicken primordial germ cell transfection methods, including the following transfection steps: S1, the separation of chicken primordial germ cell is carried out, and grouping culture in culture medium, S2, according to the proportion of 3.5:1 of pPB-GFP transposon plasmid and transposase plasmid mPB dosage Two are mixed, S3, first dilute plasmid with opti-MEN, then dilute transfection reagent with opti-MEN, plasmid diluent and transfection reagent diluent are mixed to obtain compound, the transfection operation of chicken primordial germ cell is carried out by the culture medium of different serum adding amount and adding time in the application, the influence of different serum adding amount and different serum adding time on chicken primordial germ cell transfection efficiency can be understood, then the increase and decrease of the fluorescence brightness in chicken primordial germ cell after transfection are recorded, the growth of chicken primordial germ cell after transfection is understood, so that the most suitable culture medium can be selected, serum adding condition is optimized, and more efficient chicken primordial germ cell transfection can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell transfection, in particular to a chicken primordial germ cell transfection method. BACKGROUND

[0002] Chickens have been widely used in early embryogenesis, toxicology and stem cell research, especially in avian transgenic and genome editing research. Primordial germ cells (PGCs) belong to embryonic stem cells and are the progenitor cells of spermatogonia and oogonia. They can transmit genetic information to the next generation. After in vitro culture and genetic modification, PGCs still maintain their biological characteristics. After being transplanted back into the chicken embryo blood system, they can migrate to the gonads and develop into functional gametes, producing transgenic offspring. In recent years, the development of related technologies has made it possible to develop genome-edited avian models. In the process of using chicken PGCs as a carrier for genetic manipulation, steps such as gene cloning, cell transfection, and PGCs transplantation are required, and cell transfection technology is an important part of this process.

[0003] However, the timing of serum addition in the transfection process of chicken primordial germ cells is uncertain, resulting in unstable transfection efficiency. Therefore, it is necessary to study the addition of serum to overcome the defects in the prior art. SUMMARY

[0004] The present application provides a chicken primordial germ cell transfection method, which can effectively solve the problem of the uncertain effect of serum addition time on cell transfection efficiency in the transfection process of chicken primordial germ cells, resulting in unstable transfection efficiency.

[0005] To achieve the above purpose, the present application provides the following technical scheme: a chicken primordial germ cell transfection method, comprising the following transfection steps:

[0006] S1, separating chicken primordial germ cells and culturing them in groups in a culture medium;

[0007] S2, mixing pPB-GFP transposon plasmid and transposase plasmid mPB according to a ratio of 3.5:1;

[0008] S3, first diluting the plasmid with opti-MEN, then diluting the transfection reagent with opti-MEN, mixing the plasmid diluent and the transfection reagent diluent to obtain a complex;

[0009] S4, according to the ratio of 3.5 μg of transposon plasmid, 4 μL of transfection reagent, and 3×10 3 S4, according to the ratio of 3.5 μg of transposon plasmid, 4 μL of transfection reagent, and 3×10

[0010] S5, after the beginning of transfection, serum is added to part of the culture medium, and the time for selecting is detected for the transfection result.

[0011] According to the technical scheme, the specific operation of S1 is that the chicken primordial germ cells are obtained by separating the chicken embryo gonad incubated for 5.5 days;

[0012] After separation, inoculation is carried out on the culture medium, and the culture condition is 37℃, 5% CO2, and the culture solution is replaced every 3 days;

[0013] The culture medium is provided with 4 groups, and each group of culture medium is provided with 3-5 groups:

[0014] The first group of culture medium does not add serum, and is a blank control group;

[0015] The second group of culture medium always has serum;

[0016] The third group of culture medium adds serum 1-4h after the beginning of transfection;

[0017] The fourth group of culture medium adds serum 5h after the beginning of transfection;

[0018] Among them, the serum amount added in the third group and the fourth group of culture medium is equal.

[0019] According to the technical scheme, the preparation steps of the feeder layer of the second group of culture medium in S1 are as follows: the recovered rat liver cells are cultured at 37℃, 5% CO2 until the cell density is more than 90%, the cells are digested with 2mL of trypsin with a concentration of 0.01ng / mL for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and the cobalt source is irradiated at 160 gray / min for 12min, thereby obtaining the BRL feeder layer;

[0020] The composition of the culture solution is: 1L of culture solution contains KO-DMEM 550mL, rat liver cell culture solution 300mL, fetal bovine serum 75mL, chicken serum 25mL, non-essential amino acids 10mL, GlutaMAXTM culture medium 10mL, pyruvic acid 10mL, β-mercaptoethanol 10mL, penicillin streptomycin 10mL, recombinant mouse stem cell factor 4mg, and recombinant human basic fibroblast growth factor 2.5mg;

[0021] The first group of culture medium does not add fetal bovine serum and chicken serum;

[0022] In the third group and the fourth group of culture medium, the fetal bovine serum and the chicken serum are added after the beginning of transfection.

[0023] According to the technical scheme, in S3, when the plasmid is diluted with opti-MEN, the mass volume ratio of the plasmid to opti-MEN is (1.5-3μg):150μL;

[0024] When diluting the transfection reagent with opti-MEN, the volume ratio of the transfection reagent to opti-MEN is (2-4):150.

[0025] According to the technical scheme, in the S2-S4, the transfection time is 5h.

[0026] The transfection reagent is Lipofectamine3000.

[0027] Before transfection, the chicken primordial germ cells are round, bright at the edge, and the diameter is 11-13um, and part of the cells are connected together.

[0028] The cell density is 2-3 layers, and the confluence of the uppermost layer is 100%.

[0029] The transposon plasmid is a piggyBac transposon plasmid carrying GFP protein.

[0030] According to the technical scheme, in the S5, at the beginning of transfection, the distribution of cells with fluorescent markers, i.e.

[0031] According to the technical scheme, in the S5, at the beginning of transfection, the distribution of cells with fluorescent markers, i.e.

[0032] According to the technical scheme, in the S5, the distribution of cells with fluorescent markers, i.e.

[0033] According to the technical scheme, in the S5, the intensity of the piggyBac transposon plasmid carrying GFP protein is compared with the fluorescence intensity of the previous detection, and "+" is used to represent the increase of the fluorescence intensity, and "-" is used to represent the decrease of the fluorescence intensity.

[0034] Compared with the prior art, the beneficial effects of the present application are:

[0035] 1. By setting multiple groups of culture medium during culture, and setting different serum contents and different serum adding times in different culture medium, the influence of serum on chicken primordial germ cell transfection can be experimented, the influence of serum on cell transfection can be analyzed more carefully, and a chicken primordial germ cell transfection method with better effect and higher efficiency can be explored.

[0036] 2. By using fluorescence labeling, the fluorescence of chicken primordial germ cells in the culture medium after transfection is detected, the distribution of fluorescence labeling is determined, the detection time is recorded, the proportion of fluorescence labeled cells in chicken primordial germ cells is used to represent the transfection result, and the transfection efficiency can be seen by combining time, so that the transfection situation can be understood intuitively.

[0037] 3. By detecting the fluorescence brightness of chicken primordial germ cells after transfection, the overall growth of chicken primordial germ cells after transfection can be represented, so that the subsequent situation of transfection can be understood, the transfection efficiency can be understood, the transfection of chicken primordial germ cells can be researched more comprehensively, and chicken germ cells with fast and good growth after transfection can be obtained.

[0038] In summary, by setting culture medium with different serum adding amounts and adding times for chicken primordial germ cell transfection operation, the influence of different serum adding amounts and different serum adding times on the transfection efficiency of chicken primordial germ cells can be understood, the increase and decrease of fluorescence brightness of chicken primordial germ cells after transfection is recorded, the growth of chicken primordial germ cells after transfection is understood, so that the most suitable culture medium can be selected, the serum adding conditions can be optimized, and more efficient chicken primordial germ cell transfection can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0039] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, and are used to explain the present application together with embodiments of the present application, and do not constitute a limitation on the present application.

[0040] In the drawings:

[0041] Figure 1 is a transfection step flow chart of the present application;

[0042] Figure 2 is a PGCs cell proportion fold line graph in which fluorescence is detected in embodiment 1 of the present application;

[0043] Figure 3 is a PGCs fluorescence brightness change fold line graph in embodiment 1 of the present application. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, it should be understood that the preferred embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0045] Embodiment: As shown in the figure, the present application provides a technical solution, a chicken primordial germ cell transfection method, comprising the following transfection steps: Figure 1

[0046] S1, separate the chicken primordial germ cells and group culture in the culture medium;

[0047] S2, mix the pPB-GFP transposon plasmid and the transposase plasmid mPB according to the proportion of 3.5:1;

[0048] S3, first dilute the plasmid with opti-MEN, then dilute the transfection reagent with opti-MEN, mix the plasmid diluent and the transfection reagent diluent to get a complex;

[0049] S4, according to the proportion of 3.5 μg:4 μL:3×10 3 6 of the transposon plasmid, the transfection reagent and the chicken primordial germ cells, add the complex in S3 to the chicken primordial germ cells for transfection;

[0050] S5, after the start of transfection, add serum to part of the culture medium, and select the time to detect the transfection result.

[0051] According to the above technical solution, the specific operation of S1 is: the chicken primordial germ cells are obtained by separating the chicken embryonic gonad from 5.5d incubation;

[0052] After separation, inoculate into the culture medium for culture, and the culture conditions are 37℃, 5% CO2, and the culture solution is replaced every 3d;

[0053] The culture medium is provided with 4 groups, and each group of culture medium is provided with 3 groups:

[0054] The first group of culture medium does not add serum, which is the blank control group;

[0055] The second group of culture medium always has serum;

[0056] The third group of culture medium adds serum 2.5h after the start of transfection;

[0057] The fourth group of culture medium adds serum 5h after the start of transfection;

[0058] Respectively set no serum, whole process serum, transfection serum and transfection serum, directly set the serum adding condition in different processes of transfection, so as to help the subsequent research of the influence of serum on transfection and provide experimental basis for the subsequent research;

[0059] ​The third group and the fourth group of culture media are added with equal amounts of serum.

[0060] According to the technical solution, the preparation of the feeder layer of the second group of culture media in S1 is as follows: the recovered rat liver cells are cultured at 37 DEG C and 5% CO2 until the cell density is more than 90%, the cells are digested with 2mL of trypsin with a concentration of 0.01ng / mL for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and the cells are irradiated with a cobalt source at 160 gray / min for 12min to obtain the BRL feeder layer.

[0061] The culture solution comprises 550mL of KO-DMEM, 300mL of rat liver cell culture solution, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acids, 10mL of GlutaMAXTM culture medium, 10mL of pyruvic acid, 10mL of beta-mercaptoethanol, 10mL of penicillin and streptomycin, 4mg of recombinant mouse stem cell factor, and 2.5mg of recombinant human basic fibroblast growth factor per 1L of culture solution.

[0062] The first group of culture media does not add fetal bovine serum and chicken serum.

[0063] In the third group and the fourth group of culture media, the fetal bovine serum and the chicken serum are added after the transfection starts.

[0064] According to the technical solution, when the plasmid is diluted with opti-MEN in S3, the mass-volume ratio of the plasmid to opti-MEN is 2ug:150ul.

[0065] When the transfection reagent is diluted with opti-MEN, the volume ratio of the transfection reagent to opti-MEN is 3:150.

[0066] According to the technical solution, the transfection time is 5h in S2-S4.

[0067] The transfection reagent is Lipofectamine3000.

[0068] Before the transfection, the chicken primordial germ cells are round, bright at the edge, and 12um in diameter, and part of the cells are connected together.

[0069] The cell density is 2 layers, and the confluence of the uppermost layer is 100%.

[0070] The transposon plasmid is a piggyBac transposon plasmid carrying GFP protein.

[0071] According to the above technical solution, in S5, at the beginning of transfection, observation under a fluorescence microscope is performed every 0.5 hours to observe and record the cell distribution of the piggyBac transposon plasmid carrying GFP protein in chicken primordial germ cells until the transfection ends after 5 hours.

[0072] At the beginning of transfection, each group of culture medium was placed under a fluorescence microscope for observation every 6 hours. Fluorescence microscopy was performed on chicken primordial germ cells. After the first observation of the piggyBac transposon plasmid carrying GFP protein in chicken primordial germ cells, observation and recording were performed every 2 hours. The observation and recording included the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

[0073] The distribution of the piggyBac transposon plasmid carrying the GFP protein is represented by the proportion of chicken primordial germ cells containing the piggyBac transposon plasmid carrying the GFP protein to the total number of chicken primordial germ cells. By detecting the proportion of fluorescent cells, the proportion of transfected cells can be determined, thus providing a direct understanding of the transfection status.

[0074] The intensity of the piggyBac transposon plasmid carrying the GFP protein is measured by comparing the fluorescence intensity of the current test with that of the previous test. "+" indicates an increase in fluorescence intensity, and "-" indicates a decrease in fluorescence intensity. The increase or decrease in fluorescence intensity indicates the growth of chicken primordial germ cells after transfection, providing a more comprehensive understanding of the cell condition after transfection.

[0075] like Figure 2 The graph shows the proportion of cells in PGCs that detected fluorescence.

[0076] In the first to fourth groups of culture media, the piggyBac transposon plasmid carrying the GFP protein was detected in all chicken primordial germ cells using fluorescence microscopy. Furthermore, the proportion of chicken primordial germ cells with fluorescent labels increased with time.

[0077] In the fourth culture medium, a higher proportion of cells showed fluorescence and exhibited stable growth.

[0078] In the third culture medium, the proportion of fluorescent cells detected was lower than that in the fourth culture medium, and it also showed a stable increase.

[0079] In the second group of culture medium, the proportion of cells that detected fluorescence increased faster in the early stage than in the third group, but slower in the later stage, and the proportion of cells that detected fluorescence was lower than in the third group.

[0080] The proportion of cells emitting fluorescence in the first group of culture media is less than that in the second, third and fourth groups of culture media, and grows slowly;

[0081] Therefore, the fourth group of culture media has the highest proportion of cells emitting fluorescence, indicating that the piggyBac transposon plasmid carrying the GFP protein has high transfection efficiency in chicken primordial germ cells, making it the most suitable culture medium;

[0082] As Figure 3 The line graph of the change in fluorescence intensity in PGCs is shown in the following figure:

[0083] From the first group to the fourth group of culture media, the intensity of fluorescence increases, indicating that the piggyBac transposon plasmid carrying the GFP protein grows well in chicken primordial germ cells and can achieve the expected effect. As time increases, the fluorescence intensity in chicken primordial germ cells also increases, wherein:

[0084] In the fourth group of culture media, the fluorescence intensity first increases continuously to a certain brightness and then remains stable;

[0085] In the third group of culture media, the fluorescence intensity increases intermittently to the same brightness as the fourth group and then remains stable;

[0086] In the second group of culture media, the fluorescence intensity increases intermittently and then remains stable, with a brightness lower than that in the third and fourth groups of culture media;

[0087] In the first group of culture media, the fluorescence intensity increases intermittently and then remains stable, with a brightness consistent with that in the second group of culture media;

[0088] Through comparison of the fluorescence intensity in the four groups of culture media, it can be seen that in the fourth group of culture media, the fluorescence intensity first increases to a certain brightness and then remains stable, with the shortest time and the highest efficiency, making it the most suitable culture medium.

[0089] Based on the summary of the proportion of fluorescent cells and the change in fluorescence intensity in the four groups of culture media, all four groups of culture media can achieve transfection of chicken primordial germ cells. In the fourth group of culture media, serum is added 5h after transfection. During cell transfection, the serum infection is reduced, and after transfection, the serum provides nutrition to help cell growth, resulting in the best cell transfection effect and the highest transfection efficiency, making it the most suitable culture medium.

[0090] Example 2

[0091] The specific operation of S1 is to isolate chicken primordial germ cells from chicken embryos at 5.5d of incubation;

[0092] After isolation, inoculate into culture medium and culture under the following conditions: 37℃, 5% CO2, and replace the culture medium every 3d.

[0093] The culture medium is provided with 4 groups, and each group of culture medium is provided with 4:

[0094] The first group of culture medium does not add serum, which is a blank control group;

[0095] The second group of culture medium always has serum;

[0096] The third group of culture medium adds serum 1h after the start of transfection;

[0097] The fourth group of culture medium adds serum 5h after the start of transfection;

[0098] By not adding serum, adding serum throughout the process, adding serum in the middle of transfection and adding serum after transfection, the serum addition in different processes of transfection is directly set, so as to provide experimental basis for subsequent research on the influence of serum on transfection, and facilitate subsequent research;

[0099] Among them, the serum amount of the third group and the fourth group of culture medium is equal.

[0100] According to the above technical scheme, the preparation steps of the feeder layer of the second group of culture medium in S1 are as follows: the recovered rat liver cells are cultured at 37℃, 5% CO2 to a cell density of more than 90%, the cells are digested with 2mL of trypsin with a concentration of 0.01ng / mL for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and the cells are irradiated with a cobalt source at 160 gray / min for 12min, to obtain a BRL feeder layer;

[0101] The composition of the culture solution is: 1L of culture solution contains KO-DMEM 550mL, rat liver cell culture solution 300mL, fetal bovine serum 75mL, chicken serum 25mL, non-essential amino acids 10mL, GlutaMAXTM medium 10mL, pyruvic acid 10mL, β-mercaptoethanol 10mL, penicillin streptomycin 10mL, recombinant mouse stem cell factor 4mg and recombinant human basic fibroblast growth factor 2.5mg;

[0102] No fetal bovine serum and chicken serum is added in the first group of culture medium;

[0103] In the third group and the fourth group of culture medium, fetal bovine serum and chicken serum are added after the start of transfection.

[0104] According to the above technical scheme, in S3, when the plasmid is diluted with opti-MEN, the mass volume ratio of the plasmid to opti-MEN is 1.5μg:150μL;

[0105] When the transfection reagent is diluted with opti-MEN, the volume ratio of the transfection reagent to opti-MEN is 2:150.

[0106] According to the technical scheme, in S2-S4, the transfection time is 5h;

[0107] The transfection reagent is Lipofectamine3000;

[0108] The chicken primordial germ cells before transfection are round, with bright edges, and the diameter is 11μm, and part of the cells are connected together;

[0109] The cell density is 2 layers, and the confluence of the uppermost layer is 100%;

[0110] The transposon plasmid is a piggyBac transposon plasmid carrying GFP protein.

[0111] According to the technical scheme, in S5, at the beginning of transfection, the distribution of cells with fluorescent markers, i.e., piggyBac transposon plasmids carrying GFP protein, in chicken primordial germ cells is observed under a fluorescence microscope every 0.25h until the end of transfection after 5h;

[0112] At the beginning of transfection, each group of culture medium is placed under a fluorescence microscope every 2h for observation. After the piggyBac transposon plasmid carrying GFP protein is first observed in chicken primordial germ cells, the observation is recorded every 1h. The observation records include the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

[0113] The distribution of the piggyBac transposon plasmid carrying GFP protein is represented by the ratio of the number of chicken primordial germ cells containing the piggyBac transposon plasmid carrying GFP protein to the total number of chicken primordial germ cells. The proportion of fluorescent cells is detected to determine the proportion of transfected cells, thereby intuitively understanding the transfection situation.

[0114] The intensity of the piggyBac transposon plasmid carrying GFP protein is compared with the fluorescence intensity of the previous detection. "+" is used to represent the increase in fluorescence intensity, and "-" is used to represent the decrease in fluorescence intensity. The increase and decrease in fluorescence intensity represent the growth of chicken primordial germ cells after transfection, and the situation of the cells after transfection is more fully understood.

[0115] Example 3

[0116] The specific operation of S1 is to obtain chicken primordial germ cells from the separation of chicken embryos at 5.5d;

[0117] After separation, inoculate into culture medium and culture under the condition of 37℃, 5%CO2, and replace the culture medium every 3d;

[0118] The culture medium is provided with 4 groups, and each group of culture medium is provided with 5:

[0119] The first group of culture medium does not add serum, and is a blank control group;

[0120] The second group of culture medium always has serum;

[0121] The third group of culture medium adds serum 4h after the start of transfection;

[0122] The fourth group of culture medium adds serum 5h after the start of transfection;

[0123] The serum is not added, the serum is added throughout the process, the serum is added in the middle of transfection, and the serum is added after transfection, respectively, so that the serum addition in different processes of transfection is directly set, thereby providing experimental basis for subsequent research on the influence of serum on transfection, and facilitating subsequent research;

[0124] Among them, the serum amount of the third group and the fourth group of culture medium is equal.

[0125] According to the above technical scheme, the preparation steps of the feeder layer of the second group of culture medium in S1 are as follows: the recovered rat liver cells are cultured at 37℃, 5% CO2 to a cell density of more than 90%, the cells are digested with 2mL of trypsin with a concentration of 0.01ng / mL for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and the cells are irradiated with a cobalt source at 160 gray / min for 12min, thereby obtaining a BRL feeder layer;

[0126] The composition of the culture solution is: 1L of culture solution contains KO-DMEM 550mL, rat liver cell culture solution 300mL, fetal bovine serum 75mL, chicken serum 25mL, non-essential amino acids 10mL, GlutaMAXTM culture medium 10mL, pyruvic acid 10mL, β-mercaptoethanol 10mL, penicillin streptomycin 10mL, recombinant mouse stem cell factor 4mg, and recombinant human basic fibroblast growth factor 2.5mg;

[0127] No fetal bovine serum and chicken serum is added in the first group of culture medium;

[0128] In the third group and the fourth group of culture medium, fetal bovine serum and chicken serum are added after the start of transfection.

[0129] According to the above technical scheme, in S3, when the plasmid is diluted with opti-MEN, the mass volume ratio of the plasmid to opti-MEN is 3μg:150μL;

[0130] When the transfection reagent is diluted with opti-MEN, the volume ratio of the transfection reagent to opti-MEN is 4:150.

[0131] According to the above technical scheme, in S2-S4, the transfection time is 5h;

[0132] The transfection reagent is Lipofectamine 3000;

[0133] Before transfection, the chicken primordial germ cells are round, with bright edges, and the diameter is 13um, and part of the cells are connected together;

[0134] The cell density is 3 layers, and the confluence of the uppermost layer is 100%;

[0135] The transposon plasmid is a piggyBac transposon plasmid carrying GFP protein.

[0136] According to the above technical scheme, in S5, at the beginning of transfection, the distribution of cells with fluorescent markers, i.e., piggyBac transposon plasmids carrying GFP protein, in chicken primordial germ cells is observed under a fluorescence microscope every 0.3h until the end of transfection after 5h;

[0137] At the beginning of transfection, each group of culture medium is placed under a fluorescence microscope every 4h for observation. The piggyBac transposon plasmids carrying GFP protein are first observed in chicken primordial germ cells under a fluorescence microscope, and then observed and recorded every 2h. The observation and recording content includes the fluorescence intensity of piggyBac transposon plasmids carrying GFP protein.

[0138] The distribution of piggyBac transposon plasmids carrying GFP protein is represented by the ratio of the number of chicken primordial germ cells containing piggyBac transposon plasmids carrying GFP protein to the total number of chicken primordial germ cells. The proportion of fluorescent cells is detected to determine the proportion of transfected cells, thereby intuitively understanding the transfection situation.

[0139] The intensity of piggyBac transposon plasmids carrying GFP protein is compared with the fluorescence intensity of the previous detection. "+" is used to represent the increase of fluorescence intensity, and "-" is used to represent the decrease of fluorescence intensity. The increase and decrease of fluorescence intensity represent the growth of chicken primordial germ cells after transfection, and the situation of cells after transfection is more fully understood.

[0140] Finally, it should be noted that the above only describes the preferred examples of the present application, and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications can be made to the technical solutions described in the foregoing embodiments, or some of the technical features thereof can be replaced equivalently, without departing from the spirit and principle of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for transfecting chicken primordial germ cells, characterized in that: The transfection steps include the following: S1. Chicken primordial germ cells were isolated and cultured in groups in a culture medium; S2. Mix the pPB-GFP transposon plasmid and the transposase plasmid mPB in a ratio of 3.5:

1. S3. First, dilute the plasmid with Opti-MEM, then dilute the transfection reagent with Opti-MEM. Mix the plasmid dilution and the transfection reagent dilution to obtain the complex. S4. Following the ratio of transposon plasmid, transfection reagent, and chicken primordial germ cells: 3.5 μg: 4 μL: 3 × 10⁻⁶ 3 The S3 complex was added to chicken primordial germ cells for transfection according to the specified ratio. S5. Five hours after the start of transfection, add serum to the culture medium and select a time to detect the transfection results.

2. The method for transfecting chicken primordial germ cells according to claim 1, characterized in that: The specific operation of S1 is as follows: chicken primordial germ cells are isolated from the gonads of chicken embryos incubated for 5.5 days; After isolation, the culture medium was inoculated and cultured at 37°C and 5% CO2, with the culture medium being changed every 3 days.

3. The method for transfecting chicken primordial germ cells according to claim 2, characterized in that: The preparation steps of the feeder layer of the second culture medium in S1 are as follows: Resuscitated rat hepatocytes are cultured at 37°C and 5% CO2 until the cell density is above 90%. The cells are digested with 2 mL of trypsin at a concentration of 0.01 ng / mL for 5 min, centrifuged at 1000 g / min for 5 min, the cells are collected, and irradiated with a cobalt source at 160 gray / min for 12 min to obtain the BRL feeder layer. The culture medium consists of the following components per 1L: 550mL KO-DMEM, 300mL rat hepatocyte culture medium, 75mL fetal bovine serum, 25mL chicken serum, 10mL non-essential amino acids, and GlutaMAX. TM The formula contained 10 mL of culture medium, 10 mL of pyruvate, 10 mL of β-mercaptoethanol, 10 mL of penicillin and streptomycin, 4 mg of recombinant mouse stem cell factor, and 2.5 mg of recombinant human basic fibroblast growth factor.

4. The method for transfecting chicken primordial germ cells according to claim 1, characterized in that: In step S3, when diluting the plasmid with Opti-MEM, the mass-to-volume ratio of the plasmid to Opti-MEM is (1.5-3 μg): 150 μL; When diluting the transfection reagent with Opti-MEM, the volume ratio of the transfection reagent to Opti-MEM is (2-4):

150.

5. The method for transfecting chicken primordial germ cells according to claim 1, characterized in that: In step S4, the transfection time is 5 hours. The transfection reagent was Lipofectamine 3000; Before transfection, chicken primordial germ cells are round with bright edges and a diameter of 11-13 μm, with some cells consisting of 2-4 cells linked together. The cell density is 2-3 layers, with the uppermost layer having 100% confluence. The transposon plasmid is the piggyBac transposon plasmid carrying the GFP protein.

6. The method for transfecting chicken primordial germ cells according to claim 2, characterized in that: In step S5, at the beginning of transfection, observations are performed under a fluorescence microscope every 0.25-0.5 hours to observe and record the cell distribution of the piggyBac transposon plasmid carrying the GFP protein in chicken primordial germ cells until the transfection ends after 5 hours.

7. The method for transfecting chicken primordial germ cells according to claim 6, characterized in that: In step S5, fluorescence microscopy is performed on chicken primordial germ cells. After the piggyBac transposon plasmid carrying GFP protein is first observed in chicken primordial germ cells, observations and records are made every 1-2 hours. The observation records include the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

8. The method for transfecting chicken primordial germ cells according to claim 6, characterized in that: In S5, the distribution of the piggyBac transposon plasmid carrying the GFP protein is represented by the proportion of chicken primordial germ cells containing the piggyBac transposon plasmid carrying the GFP protein to the total number of chicken primordial germ cells.

9. The method for transfecting chicken primordial germ cells according to claim 7, characterized in that: In S5, the intensity of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence intensity of the current detection with that of the previous detection, using "+" to indicate an increase in fluorescence intensity and "-" to indicate a decrease in fluorescence intensity.

Images