Chicken primordial germ cell transfection method

Abstract

The invention discloses a chicken primordial germ cell transfection method which comprises the following transfection steps: S1, separating chicken primordial germ cells, and culturing the chicken primordial germ cells in a culture medium in groups, S2, mixing pPB-GFP transposon plasmids and transposase plasmids mPB according to a ratio of 3.5: 1, S3, diluting the plasmids by using opti-MEN, diluting a transfection reagent by using opti-MEN, and carrying out transfection by using opti-MEN. According to the method, the transfection operation of the chicken primordial germ cells is carried out by setting culture media with different serum addition amounts and different serum addition times, so that the influence of different serum addition amounts and different serum addition times on the transfection efficiency of the chicken primordial germ cells can be known; and then the increase and decrease of the fluorescence brightness in the transfected chicken primordial germ cells are recorded, and the growth condition of the transfected chicken primordial germ cells is known, so that the most suitable culture medium can be selected, the serum adding condition can be optimized, and more efficient transfection of the chicken primordial germ cells can be realized.

Description

Technical Field

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

[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 ancestral cells of spermatogonia and oogonia. They can pass genetic information to the next generation. After in vitro culture and gene modification, PGCs still maintain their biological characteristics. After transplantation and reinjection into the chicken embryo blood system, they can migrate to the gonads and develop into functional gametes to produce transgenic offspring. The development of related technologies in recent years has made the development of genome editing bird models possible. In the process of using chicken PGCs as carriers for gene manipulation, it is necessary to go through steps such as gene cloning, cell transfection, and PGCs transplantation, and cell transfection technology is an important link in this process.

[0003] However, currently in the transfection process of chicken primordial germ cells, the effect of serum addition time on cell transfection efficiency is uncertain, resulting in unstable transfection efficiency. Therefore, it is necessary to study the serum addition situation to overcome the defects in the prior art. Summary of the invention

[0004] The present invention provides a chicken primordial germ cell transfection method, which can effectively solve the problem raised in the above background technology that, during the transfection process of chicken primordial germ cells, the effect of serum addition time on cell transfection efficiency is uncertain, resulting in unstable transfection efficiency.

[0005] To achieve the above object, the present invention provides the following technical solution: a chicken primordial germ cell transfection method, comprising the following transfection steps:

[0006] S1. Isolating chicken primordial germ cells and culturing them in groups in culture medium;

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

[0008] S3, first dilute the plasmid with opti-MEN, then dilute the transfection reagent with opti-MEN, and mix the plasmid dilution and the transfection reagent dilution to obtain a complex;

[0009] S4, according to the transposon plasmid, transfection reagent and chicken primordial germ cells 3.5μg: 4μL: 3×10 3 The complex in S3 was added into chicken primordial germ cells for transfection;

[0010] S5. After transfection begins, add serum to part of the culture medium and select a time to detect the transfection results.

[0011] According to the above technical solution, the specific operation of S1 is: obtaining chicken primordial germ cells by isolating the gonads of chicken embryos incubated to 5.5 days;

[0012] After separation, the cells were inoculated into culture medium and cultured at 37°C and 5% CO2, with the culture medium replaced every 3 days.

[0013] There are 4 groups of culture media, each with 3-5 cells:

[0014] The first group of culture medium was not supplemented with serum, which was the blank control group;

[0015] The second group of media always had serum;

[0016] The third group of culture media had serum added 1-4 h after the start of transfection;

[0017] The fourth group of culture medium was added with serum 5 h after the start of transfection;

[0018] Among them, the amount of serum added to the culture medium of the third and fourth groups was equal.

[0019] 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: culturing the resuscitated rat hepatocytes at 37°C and 5% CO2 to a cell density of more than 90%, digesting the cells with 2mL of 0.01ng / mL trypsin for 5min, centrifuging at 1000g / min for 5min, collecting the cells, and irradiating with a cobalt source at 160gray / min for 12min to obtain a BRL feeder layer;

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

[0021] In the first group, no fetal bovine serum or chicken serum was added to the culture medium;

[0022] In the third and fourth groups of culture media, fetal bovine serum and chicken serum were added after the start of transfection.

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

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

[0025] According to the above technical solution, in S2-S4, the transfection time is 5h;

[0026] The transfection reagent was Lipofectamine3000;

[0027] Before transfection, chicken primordial germ cells were round, with bright edges and a diameter of 11-13 μm, and some cells were 2-4 connected together;

[0028] The cell density was 2-3 layers, and the confluence of the top layer was 100%;

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

[0030] According to the above technical scheme, in S5, at the beginning of transfection, observation under a fluorescence microscope is performed every 0.25-0.5 hours to observe and record the cell distribution of the fluorescently labeled piggyBac transposon plasmid carrying GFP protein in chicken primordial germ cells until the transfection is completed after 5 hours.

[0031] According to the above technical scheme, in S5, at the beginning of transfection, each group of culture medium is placed under a fluorescence microscope for observation at intervals of 2h-6h, and fluorescence microscope observation is performed in chicken primordial germ cells. After the piggyBac transposon plasmid carrying GFP protein is observed in chicken primordial germ cells for the first time, observations and records are made every 1h-2h, and the content of the observation records includes the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

[0032] According to the above technical solution, in S5, the distribution of the piggyBac transposon plasmid carrying the GFP protein is represented by the ratio of the number of chicken primordial germ cells containing the piggyBac transposon plasmid carrying the GFP protein to the number of all chicken primordial germ cells.

[0033] According to the above technical solution, in S5, the strength of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence brightness of this detection with the fluorescence brightness of the previous detection, using "+" to indicate an increase in fluorescence brightness and "-" to indicate a decrease in fluorescence brightness.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. By setting up multiple groups of culture media during culture, and setting different serum contents and different serum addition times in different culture media, it is possible to experiment with the effect of serum on chicken primordial germ cell transfection, and more carefully analyze the effect of serum on cell transfection, helping to explore a better and more efficient chicken primordial germ cell transfection method.

[0036] 2. By using fluorescent labeling, the chicken primordial germ cells in the culture medium after transfection are detected for fluorescence, the distribution of fluorescent labels is determined, and the detection time is recorded. The proportion of fluorescently labeled cells in the chicken primordial germ cells is used to represent the result of transfection. The efficiency of transfection can be seen in combination with the time, so as to intuitively understand the transfection situation.

[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 indicated, so that the subsequent situation of transfection can be understood, and the transfection efficiency can be understood. A more comprehensive study on the transfection of chicken primordial germ cells can be conducted, and transfected chicken germ cells that grow fast and well can be obtained.

[0038] In summary, by setting culture media with different serum addition amounts and addition times to perform transfection operations on chicken primordial germ cells, we can understand the effects of different serum addition amounts and different serum addition times on the transfection efficiency of chicken primordial germ cells, and then record the increase and decrease in fluorescence brightness in the transfected chicken primordial germ cells to understand the growth of chicken primordial germ cells after transfection, so as to select the most suitable culture medium and optimize the serum addition conditions to achieve more efficient transfection of chicken primordial germ cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] In the attached picture:

[0041] Figure 1 is a flow chart of the transfection steps of the present invention;

[0042] Figure 2 is a line graph of the proportion of cells in which fluorescence is detected in the PGCs of Example 1 of the present invention;

[0043] Figure 3 It is a line graph showing the change in fluorescence brightness in PGCs of Example 1 of the present invention. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

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

[0046] S1. Isolating chicken primordial germ cells and culturing them in groups in culture medium;

[0047] S2, mixing the pPB-GFP transposon plasmid and the transposase plasmid mPB at a ratio of 3.5:1;

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

[0049] S4, according to the transposon plasmid, transfection reagent and chicken primordial germ cells 3.5μg: 4μL: 3×10 3 The complex in S3 was added into chicken primordial germ cells for transfection;

[0050] S5. After transfection begins, add serum to part of the culture medium and select a time to detect the transfection results.

[0051] According to the above technical scheme, the specific operation of S1 is: obtaining chicken primordial germ cells by isolating the gonads of chicken embryos incubated to 5.5 days;

[0052] After separation, the cells were inoculated into culture medium and cultured at 37°C and 5% CO2, with the culture medium replaced every 3 days.

[0053] There are 4 groups of culture media, each of which has 3:

[0054] The first group of culture medium was not supplemented with serum, which was the blank control group;

[0055] The second group of media always had serum;

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

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

[0058] The system can set up the following conditions: no serum addition, serum addition during the whole process, serum addition during the middle of transfection, and serum addition after transfection. The serum addition conditions can be set up directly in different processes of transfection, so as to provide experimental basis for subsequent research on the effect of serum on transfection and facilitate subsequent research.

[0059] Among them, the amount of serum added to the culture medium of the third and fourth groups was equal.

[0060] 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 resuscitated rat hepatocytes are cultured at 37°C and 5% CO2 until the cell density is above 90%, the cells are digested with 2mL of 0.01ng / mL trypsin for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and irradiated with a cobalt source at 160gray / min for 12min to obtain the BRL feeder layer;

[0061] The composition of the culture medium is as follows: each 1L of culture medium contains 550mL of KO-DMEM, 300mL of rat hepatocyte culture medium, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acids, 10mL of GlutaMAXTM medium, 10mL of pyruvic acid, 10mL of β-mercaptoethanol, 10mL of penicillin-streptomycin, 4mg of recombinant mouse stem cell factor, and 2.5mg of recombinant human basic fibroblast growth factor;

[0062] In the first group, no fetal bovine serum or chicken serum was added to the culture medium;

[0063] In the third and fourth groups of culture media, fetal bovine serum and chicken serum were added after the start of transfection.

[0064] According to the above technical scheme, in S3, when the plasmid was diluted with opti-MEN, the mass volume ratio of the plasmid to opti-MEN was 2 μg:150 μL;

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

[0066] According to the above technical scheme, in S2-S4, the transfection time was 5 h;

[0067] The transfection reagent was Lipofectamine3000;

[0068] Before transfection, chicken primordial germ cells were round, with bright edges and a diameter of 12 μm, and some cells were connected to each other.

[0069] The cell density was 2 layers, and the confluence of the top layer was 100%;

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

[0071] According to the above technical scheme, in S5, at the beginning of transfection, observation under a fluorescence microscope is performed every 0.5 h to observe and record the cell distribution of the piggyBac transposon plasmid with fluorescence labeling, i.e., carrying GFP protein, in the chicken primordial germ cells until the transfection is completed 5 h later;

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

[0073] The distribution of piggyBac transposon plasmid carrying GFP protein is expressed as the ratio of the number of chicken primordial germ cells containing piggyBac transposon plasmid carrying GFP protein to the number of all chicken primordial germ cells. By detecting the ratio of fluorescent cells, the ratio of transfected cells can be determined, so as to intuitively understand the transfection situation;

[0074] The strength of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence brightness of this test with that of the previous test. "+" indicates an increase in fluorescence brightness, and "-" indicates a decrease in fluorescence brightness. The increase or decrease in fluorescence brightness is used to indicate the growth of chicken primordial germ cells after transfection, providing a more complete understanding of the cell status after transfection.

[0075] like Figure 2 Line graph of the proportion of cells in which fluorescence was detected in the PGCs shown;

[0076] In the culture media of the first to fourth groups, the piggyBac transposon plasmid carrying GFP protein was detected in the chicken primordial germ cells by fluorescence microscopy, and as time went on, the proportion of chicken primordial germ cells with fluorescent markers increased, among which:

[0077] In the culture medium of group 4, the proportion of cells with detected fluorescence was higher and increased steadily;

[0078] In the third group of culture medium, the proportion of fluorescent cells detected was second only to that in the fourth group of culture medium, and also showed a steady increase;

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

[0080] In the culture medium of group 1, the proportion of cells with detected fluorescence was less, lower than that of the culture medium of groups 2, 3, and 4, and the growth was slow;

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

[0082] like Figure 3 Line graph showing changes in fluorescence brightness in PGCs;

[0083] From the first to the fourth group of culture media, the intensity of fluorescence increased, indicating that the piggyBac transposon plasmid carrying GFP protein grew well in chicken primordial germ cells and achieved the expected effect. As time went by, the fluorescence brightness detected in chicken primordial germ cells also increased, including:

[0084] In the fourth group of culture medium, the fluorescence brightness first increased continuously to a certain brightness and then remained stable;

[0085] In the culture medium of group 3, the fluorescence brightness intermittently increased to the same brightness as that of group 4, and then remained stable;

[0086] In the second group of culture media, the fluorescence brightness increased intermittently, and then remained stable, and the brightness was lower than that in the third and fourth groups of culture media;

[0087] In the first group of culture media, the fluorescence brightness increased intermittently, and then remained stable, and the brightness was consistent with that in the second group of culture media;

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

[0089] According to the summary of the changes in the proportion of fluorescent cells and fluorescence brightness in the four groups of culture media, all four groups of culture media can achieve the transfection of chicken primordial germ cells. In the fourth group of culture media, serum was added 5 hours after the start of transfection to reduce the infection of serum during cell transfection. After the transfection, the serum provided nutrients to help cell growth. The cell transfection effect was the best and the transfection efficiency was the highest, making it the most suitable culture medium.

[0090] Example 2

[0091] The specific operation of S1 is: to obtain chicken primordial germ cells from the gonads of chicken embryos incubated to 5.5 days;

[0092] After separation, the cells were inoculated into culture medium and cultured at 37°C and 5% CO2, with the culture medium replaced every 3 days.

[0093] There are 4 groups of culture media, each of which has 4:

[0094] The first group of culture medium was not supplemented with serum, which was the blank control group;

[0095] The second group of media always had serum;

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

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

[0098] The system can set up the following conditions: no serum addition, serum addition during the whole process, serum addition during the middle of transfection, and serum addition after transfection. The serum addition conditions can be set up directly in different processes of transfection, so as to provide experimental basis for subsequent research on the effect of serum on transfection and facilitate subsequent research.

[0099] Among them, the amount of serum added to the culture medium of the third and fourth groups was 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 resuscitated rat hepatocytes are cultured at 37°C and 5% CO2 until the cell density is above 90%, the cells are digested with 2mL of 0.01ng / mL trypsin for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and irradiated with a cobalt source at 160gray / min for 12min to obtain the BRL feeder layer;

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

[0102] In the first group, no fetal bovine serum or chicken serum was added to the culture medium;

[0103] In the third and fourth groups of culture media, fetal bovine serum and chicken serum were added after the start of transfection.

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

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

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

[0107] The transfection reagent was Lipofectamine3000;

[0108] Before transfection, chicken primordial germ cells were round, with bright edges and a diameter of 11 μm, and some cells were connected in pairs;

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

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

[0111] According to the above technical scheme, in S5, at the beginning of transfection, observation under a fluorescence microscope is performed every 0.25 h to observe and record the cell distribution of the piggyBac transposon plasmid with fluorescence marker, i.e., carrying GFP protein, in the chicken primordial germ cells until the transfection is completed 5 h later;

[0112] At the beginning of transfection, each group of culture medium was placed under a fluorescence microscope for observation every 2 hours, and the chicken primordial germ cells were observed under a fluorescence microscope. After the piggyBac transposon plasmid carrying GFP protein was first observed in the chicken primordial germ cells, the observation and recording were made every 1 hour. The content of the observation and recording included the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

[0113] The distribution of piggyBac transposon plasmid carrying GFP protein is expressed as the ratio of the number of chicken primordial germ cells containing piggyBac transposon plasmid carrying GFP protein to the number of all chicken primordial germ cells. By detecting the ratio of fluorescent cells, the ratio of transfected cells can be determined, so as to intuitively understand the transfection situation;

[0114] The strength of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence brightness of this test with that of the previous test. "+" indicates an increase in fluorescence brightness, and "-" indicates a decrease in fluorescence brightness. The increase or decrease in fluorescence brightness is used to indicate the growth of chicken primordial germ cells after transfection, providing a more complete understanding of the cell status after transfection.

[0115] Example 3

[0116] The specific operation of S1 is: to obtain chicken primordial germ cells from the gonads of chicken embryos incubated to 5.5 days;

[0117] After separation, the cells were inoculated into culture medium and cultured at 37°C and 5% CO2, with the culture medium replaced every 3 days.

[0118] There are 4 groups of culture media, each of which has 5:

[0119] The first group of culture medium was not supplemented with serum, which was the blank control group;

[0120] The second group of media always had serum;

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

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

[0123] The system can set up the following conditions: no serum addition, serum addition during the whole process, serum addition during the middle of transfection, and serum addition after transfection. The serum addition conditions can be set up directly in different processes of transfection, so as to provide experimental basis for subsequent research on the effect of serum on transfection and facilitate subsequent research.

[0124] Among them, the amount of serum added to the culture medium of the third and fourth groups was 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 resuscitated rat hepatocytes are cultured at 37°C and 5% CO2 until the cell density is above 90%, the cells are digested with 2mL of 0.01ng / mL trypsin for 5min, centrifuged at 1000g / min for 5min, the cells are collected, and irradiated with a cobalt source at 160gray / min for 12min to obtain the BRL feeder layer;

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

[0127] In the first group, no fetal bovine serum or chicken serum was added to the culture medium;

[0128] In the third and fourth groups of culture media, fetal bovine serum and chicken serum were added after the start of transfection.

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

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

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

[0132] The transfection reagent was Lipofectamine3000;

[0133] Before transfection, chicken primordial germ cells were round, with bright edges and a diameter of 13 μm, and some cells were three connected together;

[0134] The cell density was 3 layers, and the confluence of the top layer was 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, observation under a fluorescence microscope is performed every 0.3 h to observe and record the cell distribution of the piggyBac transposon plasmid with fluorescence labeling, i.e., carrying GFP protein, in the chicken primordial germ cells until the transfection is completed 5 h later;

[0137] At the beginning of transfection, each group of culture medium was placed under a fluorescence microscope for observation every 4 hours, and the chicken primordial germ cells were observed under a fluorescence microscope. After the piggyBac transposon plasmid carrying GFP protein was first observed in the chicken primordial germ cells, the observation and recording were made every 2 hours. The observation and recording contents included the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

[0138] The distribution of piggyBac transposon plasmid carrying GFP protein is expressed as the ratio of the number of chicken primordial germ cells containing piggyBac transposon plasmid carrying GFP protein to the number of all chicken primordial germ cells. By detecting the ratio of fluorescent cells, the ratio of transfected cells can be determined, so as to intuitively understand the transfection situation;

[0139] The strength of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence brightness of this test with that of the previous test. "+" indicates an increase in fluorescence brightness, and "-" indicates a decrease in fluorescence brightness. The increase or decrease in fluorescence brightness is used to indicate the growth of chicken primordial germ cells after transfection, providing a more complete understanding of the cell status after transfection.

[0140] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chicken primordial germ cell transfection method, characterized in that: The transfection steps include the following: S1. Isolating chicken primordial germ cells and culturing them in groups in culture medium; S2, mixing the pPB-GFP transposon plasmid and the transposase plasmid mPB at a ratio of 3.5:1; S3, first dilute the plasmid with opti-MEN, then dilute the transfection reagent with opti-MEN, and mix the plasmid dilution and the transfection reagent dilution to obtain a complex; S4, according to the transposon plasmid, transfection reagent and chicken primordial germ cells 3.5μg: 4μL: 3×10 3 The complex in S3 was added into chicken primordial germ cells for transfection; S5. After transfection begins, add serum to part of the culture medium and select a time to detect the transfection results.

2. A chicken primordial germ cell transfection method according to claim 1, characterized in that: The specific operation of S1 is: separating and obtaining chicken primordial germ cells from the gonads of chicken embryos incubated to 5.5 days; After separation, the cells were inoculated into culture medium and cultured at 37°C and 5% CO2, with the culture medium replaced every 3 days. The culture medium is provided in 4 groups, and each group of culture medium is provided with 3-5: The first group of culture medium was not supplemented with serum, which was the blank control group; The second group of media always had serum; The third group of culture media had serum added 1-4 h after the start of transfection; The fourth group of culture medium was added with serum 5 h after the start of transfection; Among them, the amount of serum added to the culture medium of the third and fourth groups was equal.

3. A chicken primordial germ cell transfection method according to claim 2, characterized in that: The preparation steps of the feeder layer of the second group of culture medium in S1 are as follows: culturing resuscitated rat hepatocytes at 37° C. and 5% CO2 to a cell density of more than 90%, digesting the cells with 2 mL of 0.01 ng / mL trypsin for 5 min, centrifuging at 1000 g / min for 5 min, collecting the cells, and irradiating with a cobalt source at 160 gray / min for 12 min to obtain a BRL feeder layer; The composition of the culture medium is as follows: each 1L of culture medium contains 550mL of KO-DMEM, 300mL of rat hepatocyte culture medium, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acids, 10mL of GlutaMAXTM medium, 10mL of pyruvic acid, 10mL of β-mercaptoethanol, 10mL of penicillin-streptomycin, 4mg of recombinant mouse stem cell factor, and 2.5mg of recombinant human basic fibroblast growth factor; In the first group, no fetal bovine serum or chicken serum was added to the culture medium; In the third and fourth groups of culture media, fetal bovine serum and chicken serum were added after the start of transfection.

4. A chicken primordial germ cell transfection method according to claim 1, characterized in that: In S3, when the plasmid is diluted with opti-MEN, the mass volume ratio of the plasmid to opti-MEN is (1.5-3 μg): 150 μL; When diluting the transfection reagent with opti-MEN, the volume ratio of transfection reagent to opti-MEN is (2-4):

150.

5. A chicken primordial germ cell transfection method according to claim 1, characterized in that: In S2-S4, the transfection time is 5 h; The transfection reagent was Lipofectamine3000; Before transfection, chicken primordial germ cells were round, with bright edges and a diameter of 11-13 μm, and some cells were 2-4 connected together; The cell density was 2-3 layers, and the confluence of the top layer was 100%; The transposon plasmid is a piggyBac transposon plasmid carrying GFP protein.

6. A chicken primordial germ cell transfection method according to claim 2, characterized in that: In S5, at the beginning of transfection, observation under a fluorescence microscope was performed every 0.25-0.5 h to observe and record the cell distribution of the fluorescently labeled piggyBac transposon plasmid carrying GFP protein in chicken primordial germ cells until the transfection was completed 5 h later.

7. A chicken primordial germ cell transfection method according to claim 6, characterized in that: In S5, at the beginning of transfection, each group of culture medium is placed under a fluorescence microscope for observation at intervals of 2h-6h, and fluorescence microscope observation is performed in chicken primordial germ cells. After the piggyBac transposon plasmid carrying GFP protein is first observed in the chicken primordial germ cells, observations and records are made every 1h-2h, and the content of the observation records includes the fluorescence intensity of the piggyBac transposon plasmid carrying GFP protein.

8. A chicken primordial germ cell transfection method according to claim 6, characterized in that: In S5, the distribution of the piggyBac transposon plasmid carrying the GFP protein is represented by the ratio of the number of chicken primordial germ cells containing the piggyBac transposon plasmid carrying the GFP protein to the number of all chicken primordial germ cells.

9. A chicken primordial germ cell transfection method according to claim 6, characterized in that: In S5, the strength of the piggyBac transposon plasmid carrying the GFP protein is determined by comparing the fluorescence brightness of the current test with the fluorescence brightness of the previous test, using "+" to indicate an increase in fluorescence brightness and "-" to indicate a decrease in fluorescence brightness.

Images