CN112522315A

CN112522315A - Chicken primordial germ cell transfection method

Info

Publication number: CN112522315A
Application number: CN202011445596.8A
Authority: CN
Inventors: 邹娴; 罗成龙; 舒鼎铭; 李莹; 何燕华; 何静怡
Original assignee: Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-19
Anticipated expiration: 2040-12-08
Also published as: CN112522315B

Abstract

The invention discloses a chicken primordial germ cell transfection method. Belongs to the technical field of cell transfection. The method comprises the following steps: separating and culturing chicken primordial germ cells; transfecting, wherein cells before transfecting are circular, the edges of the cells are bright, the diameter of the cells is 11-13 mu m, and 2-4 cells are connected together; the ratio of the plasmid, the transfection reagent and the number of the chicken primordial germ cells is (1.5-3 mu g): 2-4 mu L):10³～10⁴One); 2-3 layers of cells are transfected, and the confluence degree of the uppermost layer is close to 100%; the transfection medium is a complete medium with serum and double antibodies; the transfection time is 5-12 h; the transfection reagent was Lipofectamine 3000. The invention has the beneficial effects that: the transfection efficiency reached 23.4%. Lays a foundation for the application of the poultry genome editing technology.

Description

Chicken primordial germ cell transfection method

Technical Field

The invention relates to the technical field of cell transfection, in particular to a chicken primordial germ cell transfection method.

Background

Chickens have been widely used for early embryogenesis, toxicology, and stem cell studies, particularly avian transgenesis and genome editing studies. Primordial Germ Cells (PGCs) belong to embryonic stem Cells, are progenitors of spermatogonial and oogonial Cells, and can transmit genetic information to the next generation. PGCs still maintain the biological characteristics after in vitro culture and gene modification, and can migrate to the gonads and develop into functional gametes after being transplanted and reinjected into a chick embryo blood vessel system to generate transgenic offspring. In recent years, PGCs are combined into Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9(Cas9) technologies, so that the development of a new era genome editing bird model is possible, wherein the development of the bird model can be realized, and the new era genome editing bird model comprises the introduction of a LOXP locus into a chicken IgH locus, the insertion of a targeted avian ovalbumin gene and a marker gene into a Z chromosome and the anti-J subtype chicken leukemia model. In the gene operation process by using chicken PGCs as a vector, the steps of gene cloning, cell transfection, PGCs transplantation and the like are required, and the cell transfection technology is an important link in the process.

At present, chicken PGCs (PGCs) transfection technologies mainly comprise lipofection, adenovirus or lentivirus transfection and electrotransfection, wherein the lipofection technology is more applied due to safety, simple and convenient operation and low cost. However, the transfection efficiency of chicken PGCs liposome is only 5% -10%, and the reasons are mainly as follows: 1) the transfection efficiency of the embryonic stem cells is generally low; 2) the chicken PGCs are suspension cells and the culture conditions are complex; 3) lipofectamine2000, a Lipofectamine reagent, is highly toxic.

In conclusion, how to provide a chicken primordial germ cell transfection method with high transfection efficiency is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention provides a method for transfecting chicken primordial germ cells. The transfection efficiency is improved to 23.4 percent by optimizing the transfection process, and a foundation is laid for the research of efficiently preparing transgenic chickens and gene editing chickens.

In order to achieve the purpose, the invention adopts the following technical scheme:

a chicken primordial germ cell transfection method comprises the following steps:

(1) separating and culturing chicken primordial germ cells;

(2) transfection:

mixing PB transposon plasmid and transposase plasmid in a dosage ratio of 3: 1;

diluting the plasmid by using opti-MEN, wherein the mass volume ratio of the plasmid to the opti-MEN is (1.5-3 mu g) 150 mu L, diluting the transfection reagent by using the opti-MEN, wherein the volume ratio of the transfection reagent to the opti-MEN is (2-4) 150, mixing the diluted plasmid and the diluted transfection reagent, and standing at room temperature for 10-15 minutes to obtain a compound;

③ adding the compound into chicken primordial germ cells;

wherein the ratio of the plasmid, the transfection reagent and the chicken primordial germ cells is (1.5-3 mu g): (2-4 mu L):10³～10⁴One);

the transfection time is 5-12 h;

feeder cells are required for transfection;

the transfection reagent is Lipofectamine 3000.

Furthermore, the proportion of the plasmid, the transfection reagent and the chicken primordial germ cells is (1.5-3 mu g): 3-4 mu L):10³A plurality of;

the transfection time is 6 h;

the culture medium used for transfection is a complete culture medium with serum and double antibodies;

feeder cells are required for transfection;

the transfection reagent is Lipofectamine 3000.

Furthermore, the cells before transfection of the chicken primordial germ cells are round, have bright edges and diameters of 11-13 mu m, and 2-4 partial cells are connected together;

the cell density is 2-3 layers, and the confluence degree of the uppermost layer is close to 100%;

the proportion of the plasmid, the transfection reagent and the chicken primordial germ cells is 3 mug, 4 mug, 5 multiplied by 10³A plurality of;

the transfection time is 5 h;

feeder cells are required for transfection;

the transfection reagent is Lipofectamine 3000.

The beneficial effects are as follows: transfection efficiencies near 100% or less than 70% cell confluence are lower than 70% to 90% cell confluence. Thus transfection is routinely performed as a monolayer of cells with 70% to 90% cell confluence. Under the condition that the plasmid concentration and the transfection reagent dosage are the same, the method improves the density of the primordial germ cells, enables the cells to have 2-3 layers in the holes, enables the confluence degree of the uppermost layer to be close to 100%, and can improve the transfection efficiency. This is probably because chicken primordial germ cells are round suspension cells with bright edges, the density is high, the gaps among the cells are small, the contact surface of plasmid DNA and the cells is large, the resistance of entering the cells is reduced, and the transfection level is improved. Meanwhile, the transfection efficiency cannot be improved by increasing the plasmid concentration or the dosage of the transfection reagent regardless of increasing the cell density.

Further, the plasmid is a piggyBac transposon plasmid carrying GFP protein.

Further, the specific operation of the step (1) is as follows:

(11) separating chicken embryo gonad from the chicken embryo incubated to 5.5d to obtain chicken primordial germ cells;

(12) separating, inoculating to BRL feeder layer, and culturing at 37 deg.C and 5% CO₂The culture medium was changed every 2.5 days.

The beneficial effects are as follows: the invention separates and cultures PGCs from gonads of chick embryos which are hatched for 5.5 days. The advantage of using gonadal PGCs over circulating blood PGCs is that more PGCs can be obtained from a single embryo, and each embryo can more easily develop many PGCs, more efficiently selecting lines of PGCs with germ cell pluripotency to produce germline chimeras and transgenic chickens.

Further, the preparation steps of the BRL feeding layer are as follows:

resuscitating rat liver cells at 37 deg.C and 5% CO₂Culturing under the condition until the cell density is more than 90%, digesting the cells with 2mL of pancreatin with the concentration of 0.01ng/mL for 5min, centrifuging for 5min at 1000g/min, collecting the cells, and radiating for 12min by a cobalt source 160gray/min to obtain the BRL feeder layer.

Further, the culture solution comprises the following components: each 1L of culture solution contains 550mL of KO-DMEM, 300mL of rat hepatocyte culture solution, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acid, 10mL of GlutaMAXTM culture medium, 10mL of pyruvic acid, 10mL of beta-mercaptoethanol, 10mL of penicillin streptomycin, 4mg of recombinant mouse stem cell factor and 2.5mg of recombinant human basic fibroblast growth factor.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the highest transfection efficiency of the method reaches 23.4 percent by analyzing through a flow cytometer, and a foundation is laid for the application of the poultry genome editing technology.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the culture conditions of chicken PGCs isolated in example 1 of the present invention, wherein A is a morphological feature map of chicken PGGs; b is a chicken PGCs surface marker protein map, wherein green fluorescence represents the expression of SSEA-1 protein, and red fluorescence represents the expression of DAZL protein;

FIG. 2 is a diagram showing a plasmid map constructed in example 2 of the present invention;

FIG. 3 is a graph showing the results of flow cytometry sorting of positive chicken PGCs in example 6 of the present invention, wherein A is the result of sorting of negative DF1 cells that have not been transfected with any plasmid; b is the result of sorting DF1 cells transfected with GFP plasmid; c is a positive chicken PGCs sorting result under the B3 transfection condition; d is the positive chicken PGCs sorting result under the transfection condition of example 4;

FIG. 4 is a drawing showing PGCs stably expressing green fluorescent protein in example 6 of the present invention, in which A is a white light field; b is a green fluorescent field.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the embodiment of the invention:

the hatching eggs are Hui Yang beard chicken hatching eggs and come from original chicken farms of animal science research institute of agriculture academy of sciences of Guangdong province. Incubate to the desired age at 38.5 ℃ in an Rcom PRO 50 incubator (Rutox) at a relative humidity of 60%.

The PiggyBac transposon is supplied by southern china university of agriculture and contains the GFP gene (constructed in this laboratory).

The required medicament is a conventional experimental medicament purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.

Example 1 isolation and culture of chick primordial germ cells

The method comprises the following steps:

preparing a BRL (broilers) feeding layer: resuscitating rat liver cells at 37 deg.C and 5% CO₂Culturing under the condition until the cell density is more than 90%, digesting the cells with 2mL of pancreatin with the concentration of 0.01ng/mL for 5min, centrifuging for 5min at 1000g/min, collecting the cells, and radiating for 12min by a cobalt source 160gray/min to obtain the BRL feeder layer.

Preparation of culture solution: each 1L of culture solution contains 550mL of KO-DMEM, 300mL of rat hepatocyte culture solution, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acid, 10mL of GlutaMAXTM culture medium, 10mL of pyruvic acid, 10mL of beta-mercaptoethanol, 10mL of penicillin streptomycin, 4mg of recombinant mouse stem cell factor and 2.5mg of recombinant human basic fibroblast growth factor.

Separating gonad of HUIYANG HUXU JI embryo to 5.5d to obtain chicken PGCs (40 chicken embryos separated in total), inoculating to BRL feeder layer, culturing at 37 deg.C and 5% CO₂. Changing the culture solution every 2.5 days, and carrying out passage 1 time on the chicken primordial germ cells every 3-4 days.

After 3 weeks of culture, morphological characteristics of the cells were observed by an inverted microscope, and after 60 days of culture, the cell surface marker proteins were detected by immunofluorescence.

(II) results:

in 40 isolated chick embryos, 10 PGCs reached a cell count of 5.0X 10 before the end of week 3⁴It was considered that the strain was successfully established. All 10 PGCs lines were from a single embryo. The cells are round, bright in edge, large in volume, large in nucleus, etc., and usually two cells are connected together or strung to meet the morphological characteristics of PGCs (FIG. 1A).

After 60 days of PGCs culture, the expression of the surface marker proteins SSEA-1 and DAZL of PGCs was detected by immunofluorescence (FIG. 1B).

Example 2 piggyBac transposon vector construction carrying GFP protein

(1) Construction of Green fluorescent expression vector

Carrying out double enzyme digestion on the extracted plasmid VK008-01 and pCyL50 vectors by using EcoR I and Xho I enzymes respectively to obtain pGK-neo-T2A-EGFP fusion fragments; the restriction system was described in the specifications of restriction enzymes Xho I and EcoR I from Fermentas. Mixing, digesting at 37 deg.C for 15-30min, and recovering the target fragment according to E.Z.N.A Gel Extraction Kit agarose Gel recovery and purification Kit agarose Gel specification of OMEGA.

And (3) carrying out enzyme digestion on the purified pGK-neo-T2A-EGFP fusion fragment and the pCyL50 vector according to the concentration ratio of 1: 1 and 5 Xpremixed enzyme reagent, at 50 ℃ for 15min to complete the connection, specifically refer to the In-Fusion Cloning Kit (TAKARA) instructions.

(2) Ligation product transformation of competent cell DH5a

Thawing the preserved competent cell DH5 alpha in ice, taking 30 mu L of competent cell and 10 mu L of ligation product, gently mixing, carrying out ice bath for 30min, carrying out heat shock for 90s at 42 ℃, rapidly transferring to the ice bath for cooling for 2-3min, adding 1mL of LB liquid culture medium without antibiotics, shaking and culturing at 37 ℃ and 160r/min for 1h, taking 200 mu L of bacterial liquid, coating the bacterial liquid on an LA plate, and culturing overnight at 37 ℃.

(3) PCR identification of Positive colonies

Selecting a single white colony from the cultured plate, inoculating the single white colony in 1mL LB liquid culture medium containing ampicillin, shaking and culturing at 37 ℃ for 12-16h, designing a primer, directly taking a bacterial liquid as a template to carry out PCR identification, carrying out a reaction system and a program according to the instructions of Dongshan TaqMix kit, and detecting a PCR product by using 1% agarose gel electrophoresis.

(4) Plasmid extraction, identification and sequencing

The positive bacteria liquid is inoculated into 5ml LB liquid culture medium containing Amp again, and cultured for 12-16h at 37 ℃ and 220r/min by shaking, referring to the instruction provided by E.Z.N.A.TM.plasmid Mini Kit I of OMEGA.

Taking 1 mu L of extracted plasmid, selecting exogenous fragments to insert into the restriction enzyme sites Xho I and EcoR I on both sides for restriction enzyme identification, and detecting the restriction enzyme result by electrophoresis. When the enzyme cutting result is consistent with the expectation, 2-3 clones are selected and sent to Shanghai Yingjun biotechnology service company for forward and reverse direction sequencing, and the sequencing result and the target sequence are analyzed.

(5) Preparation of plasmid for transfection

Carrying out amplification culture on a bacterial solution of a recombinant plasmid (p-PB-neoEGFP) with correct sequencing at 37 ℃ for 12-16h at 220r/min, collecting thalli and carrying out endotoxin-removing plasmid extraction. The procedure was performed according to the OMEGA E.Z.N.A.TM.EndoFee Plasmid Mini Kit II reagent instructions.

The extracted plasmid is cut by restriction enzyme sites EcoR I and Asc I, 5 mu L of cut product and 1 mu L of plasmid DNA are taken for agarose gel electrophoresis, and the correctness and the plasmid conformation are identified. And 2 u L plasmid, using the spectrophotometer determination of OD260 and OD280 optical density absorption value, using the R value ═ OD260/OD280 estimation of plasmid DNA purity. The plasmid DNA with the R value of 1.8-2.0 and the supercoiled conformation of more than 70 percent can be used for cell transfection experiments.

The constructed plasmid map is shown in FIG. 2.

Example 3 transfection

Step (one)

(1) Chicken PGCs were transferred to 24-well plates and inoculated 10 per well prior to transfection³One cell, at which the cell density is only one layer, with a degree of confluence of about 80%.

(2) The piggyBac transposon plasmid carrying the GFP protein prepared in example 2 was transfected with 3 different transfection reagents, respectively: lipofectamine2000 (denoted by A), Lipofectamine 3000 (denoted by B), and Lipofectamine LTX & Plus Reagent (denoted by C). And different plasmid dosage and transfection reagent dosage are set. The specific experimental groups are shown in table 1.

TABLE 1 Experimental groups

(3) The transfection procedure was performed. Specifically, serum-free, double-antibody-free PGCs were used for transfection. Collecting cells after 6h of transfection, centrifuging, re-suspending, inoculating on new feeder layer, adding fresh PGCs culture solution, standing at 37 deg.C and 5% CO₂After incubation in the incubator for a further 48h, the images were photographed using an inverted microscope and analyzed. Each set was 3 replicates.

(II) results

Specific results are shown in table 2.

TABLE 2 number of positive cells in different groups (number/well)

Note: different letters in the same column indicate significant P < 0.05.

The results in Table 2 show that the Lipofectamine 3000 transfection reagent has the highest number of B3 positive cells, and the number of cells per well is about 2X 10 as calculated by a cytometer³It was found that the transfection efficiency was about 13.9%, which was highIn all groups; the second was Lipofectamine 3000 transfection reagent B2, transfection efficiency was about 5.1%, and the third was Lipofectamine LTX&The transfection efficiency of Plus Reagent C3 was about 4.0%. In the experimental process, the Lipofectamine2000 transfection reagent group and high-concentration groups B4 and C4 of the other two reagents have large damage to chicken PGCs, and a large amount of chicken PGCs are dead after 48 hours.

Example 4 transfection

Step (one)

(2) The transfection efficiency was measured by extracting cells 48h after transfection in the transfection step using the transfection reagent Lipofectamine 3000. Transfecting the piggyBac transposon plasmid carrying GFP with Lipofectamine 3000 under the transfection condition of B3 in example 3 for 5h, 24h and 48h, collecting the cells, centrifuging, inoculating the cells onto a new feeder layer after resuspension, adding a fresh PGCs culture solution, placing the culture solution at 37 ℃ and 5% CO₂And continuing culturing in the incubator. After 24 and 48h of transfection, photographs were taken with an inverted microscope and analyzed. Each set was 3 replicates.

(II) results

The transfection time was 24h and 48h before the collection of the groups of cells, which appeared to be heavily adherent and died. The transfection time was 5h for the cell collection group, which was not observed.

Example 5 transfection

Step (one)

(1) Chicken PGCs were transferred to 24-well plates at 5X 10 inoculum size prior to transfection³And (4) one cell per well, wherein the cell density is 2-3 layers, and the confluence degree of the cells on the uppermost layer is close to 100%.

(2) Transfecting a piggyBac transposon plasmid carrying GFP (green fluorescent protein) by using Lipofectamine 3000, wherein the using amount of the plasmid is 3 mu g, the using amount of the Lipofectamine 3000 is 4 mu L, transfecting by using a PGCs complete culture medium with serum and double antibody for 5h and 12h, collecting cells, centrifuging, inoculating to a new feeder layer after resuspension, adding a fresh PGCs culture solution, placing at 37 ℃ and 5% CO₂IncubatorAfter further incubation for 48h, the images were photographed with an inverted microscope and analyzed. Each set was 3 replicates.

(II) results

The number of positive cells in each well exceeds 1000, and the number of cells in each well is about 7X 10 as calculated by a cell counter³In addition, the transfection efficiency was more than 14.2%, which is higher than that of the group B3 in which the transfection efficiency was the highest in example 3.

Example 6 flow cytometry sorting of PGCs stably expressing GFP proteins

(1) Subject: firstly, chicken PGCs transfected under the transfection condition of B3 in example 3; ② chicken PGCs transfected under the transfection conditions in example 5; ③ taking DF1 cells transfected with GFP plasmid as positive control; and fourthly, DF1 cells which are not transfected with any plasmid are used as negative control. The transfection time was 5h for all groups.

(2) The experimental steps are as follows: after transfection, culture was continued for 72 hours, and positive cells were sorted by flow cytometry using GFP luminescent gene. Before machine sorting, a sterile working solution (with the components distilled water and 75% ethanol) was prepared to wash the flow cytometer (BD FACSAriaII). Collecting chicken PGCs and DF1 cells into a 15mL centrifuge tube, and suspending the cells with PBS, wherein the cell number is more than 10⁶one/mL. Positive control and negative control are used for setting sampling parameters and the range of a cross gate of the flow cytometer respectively, and then chicken PGCs are sorted.

(4) The experimental results are as follows: as shown in fig. 3.

As shown in fig. 3, the proportion of positive cells in the DF1 negative control group was 0%, the proportion of positive cells in the DF1 positive control group was 46.2%, the proportion of positive chicken PGCs under the B3 transfection condition was 12.7%, and the proportion of positive chicken PGCs under the example 5 transfection condition was 23.4%.

The positive PGCs sorted by the flow cytometer were inoculated into a 24-well plate and cultured, and the state was good (fig. 4). After further culturing for 72 hours, subculture was carried out.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A chicken primordial germ cell transfection method is characterized by comprising the following steps:

(1) separating and culturing chicken primordial germ cells;

(2) transfection:

mixing PB transposon plasmid and transposase plasmid in a dosage ratio of 3: 1;

③ adding the compound into chicken primordial germ cells;

the proportion of the plasmid, the transfection reagent and the chicken primordial germ cells is (1.5-3 mu g): 2-4 mu L):10³～10⁴One);

wherein the transfection time is 5-12 h;

the transfection reagent is Lipofectamine 3000.

2. The method of claim 1, wherein the ratio of the plasmid to the transfection reagent to the chicken primordial germ cells is (1.5-3 μ g) to (3-4 μ L) 10³A plurality of;

the transfection time is 6 h;

the transfection reagent is Lipofectamine 3000.

3. The method for transfecting chicken primordial germ cells as claimed in claim 1, wherein the cells before transfection of the chicken primordial germ cells are round, bright in edge, 11-13 μm in diameter, and 2-4 partial cells are connected together;

the transfection time is 5 h;

the transfection reagent is Lipofectamine 3000.

4. The method for transfecting chicken primordial germ cells as recited in any of claims 1 to 3, wherein said plasmid is a piggyBac transposon plasmid carrying GFP.

5. The method for transfecting chicken primordial germ cells as claimed in any of claims 1 to 3, wherein the step (1) is specifically performed by:

6. The method of claim 5, wherein the BRL feeder layer is prepared by:

7. The method of claim 5, wherein the culture medium comprises: each 1L of culture solution contains 550mL of KO-DMEM, 300mL of rat hepatocyte culture solution, 75mL of fetal bovine serum, 25mL of chicken serum, 10mL of non-essential amino acid, 10mL of GlutaMAXTM culture medium, 10mL of pyruvic acid, 10mL of beta-mercaptoethanol, 10mL of penicillin streptomycin, 4mg of recombinant mouse stem cell factor and 2.5mg of recombinant human basic fibroblast growth factor.