A method for transfecting fibroblast cells
By injecting genomic DNA into the nucleus of porcine fetal fibroblasts using a micromanipulator, the problem of unstable cell transfection in existing technologies has been solved, achieving efficient and stable transfection and obtaining cell lines without selection markers.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
- Filing Date
- 2023-04-21
- Publication Date
- 2026-06-30
AI Technical Summary
Current electroporation methods cannot guarantee that all successfully transfected cells are stably transfected, resulting in cloned pigs carrying selection markers that cannot be propagated.
Genomic DNA was directly injected into the nucleus of porcine fetal fibroblasts using a micromanipulator. The injection needle angle was adjusted to 30–60°, and cell suspension droplet incubation was performed in a specific incubation environment. The gene plasmid concentration was controlled at 150–200 ng/μL to achieve efficient and stable transfection.
A stable transfection efficiency of 92.3% was achieved, resulting in cell lines without selection markers, thus avoiding further selection steps.
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Figure CN116287004B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell transfection technology, and in particular to a method for gene transfection of fibroblasts. Background Technology
[0002] Somatic cell cloned pigs possess significant advantages and application potential in human animal disease models and xenotransplantation, making them a current hot topic in somatic cell cloning research. The organic combination of somatic cell cloning technology and transgenic technology has greatly improved the efficiency of livestock transgenic processes, making the production of transgenic animals using cloning technology a reality. The application of somatic cell cloning transgenic technology in livestock production can accelerate the process of pig genetic improvement, leading to the development of superior pig breeds.
[0003] Currently, people have successfully obtained cloned pigs using porcine fetal fibroblasts, kidney cells, granulosa cells, ear tissue cells, and skin fibroblasts. Due to the characteristics of porcine fetal fibroblasts, such as long lifespan in vitro, ease of culture, and ability to withstand a certain degree of genetic screening, they have become the main donor cells for somatic cell cloned transgenic pigs.
[0004] Cell transfection is a specialized technique for introducing exogenous molecules such as DNA and RNA into eukaryotic cells. With the deepening of gene and protein function research, transfection has become a fundamental method frequently encountered in laboratory work. Cell transfection can be broadly classified into three pathways: physical-mediated, chemical-mediated, and biological-mediated. Electroporation has become the mainstream method in laboratories due to its convenience, simplicity, minimal influencing factors, and low cell selectivity. However, electroporation primarily uses a high-intensity electric field to momentarily increase cell membrane permeability, thereby absorbing exogenous molecules from the surrounding medium and introducing DNA and RNA into prokaryotic and eukaryotic cells. However, this method cannot guarantee 100% cell transfection success, nor can it guarantee that all successfully transfected cells are stably transfected. Therefore, to obtain the desired cell line, this method can only introduce exogenous genes carrying selection markers, resulting in cell lines with selection markers. Somatic cell clones of transgenic pigs obtained through somatic cell nuclear transfer from these selected cells also carry selection markers, preventing the propagation of this pig breed into a population.
[0005] Therefore, it is urgent to develop a more effective transfection technology that can import transfections without screening markers and achieve high-efficiency and stable transformation. Summary of the Invention
[0006] The purpose of this invention is to provide a method for gene transfection of fibroblasts. This method involves directly injecting the genomic DNA to be introduced into the nucleus of porcine fetal fibroblasts using a micromanipulator, achieving transfection. The method provided by this invention directly injects genes into the cells to be transfected one by one using a micromanipulator, eliminating the need for further selection. Therefore, the injected gene does not need to carry selection markers, thus achieving the goal of obtaining a cell line without selection markers. Simultaneously, it enables highly efficient and stable transfection.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] This invention provides a method for gene transfection of fibroblasts, the method comprising:
[0009] A fibroblast suspension with a cell concentration of 1-2 cells / µL was obtained. Cell suspension microdroplets were prepared in a cell culture dish from the fibroblast suspension. Each cell suspension microdroplet was formed from 10±0.5µL of the fibroblast suspension.
[0010] The cell culture dish containing cell suspension droplets was placed in a microenvironment for incubation. After the cells adhered to the dish, multiple adherent fibroblasts were formed. The cells were then added to a cell culture incubator for further culture.
[0011] Obtain a plasmid solution containing the linearized target gene at a concentration of 150–200 ng / μL;
[0012] The plasmid solution containing the linearized target gene is drawn into an injection needle, and the angle between the injection needle and the horizontal plane is adjusted to 30-60°. The plasmid is then injected into each of the adherent fibroblasts in sequence. After injection, single-cell culture is performed to obtain stably transfected transgenic cell clones.
[0013] Furthermore, the cell suspension droplets are prepared in cell culture dishes with a diameter of 35 mm, with 6 ± 1 droplets prepared in each 35 mm diameter cell culture dish. During inoculation, the distance between the inoculation position of the droplet closest to the edge of the cell culture dish and the edge of the cell culture dish is 1 ± 0.2 cm.
[0014] Furthermore, the cell culture dish containing cell suspension droplets is placed in a nested microenvironment for incubation. This nested microenvironment is formed by combining multiple cell culture dishes, specifically including:
[0015] The cell culture dish containing cell suspension droplets is a 35mm diameter cell culture dish, comprising a 35mm diameter dish body and a first dish lid;
[0016] The first dish cover is covered by the second dish cover, which is the cover of a cell culture dish with a diameter of 90 mm;
[0017] The second dish cover is covered by a third dish cover, which is the cover of a cell culture dish with a diameter of 110 mm;
[0018] The bottom of the 35mm diameter dish is provided with a partition plate, and the lower end of the second dish lid is located on the partition plate;
[0019] The bottom of the isolation plate is provided with a cell culture dish with a diameter of 110 mm, and the lower end of the third dish cover is located on the cell culture dish with a diameter of 110 mm.
[0020] The second lid has one or more vent holes at its top; a water injection zone is formed between the outer wall of the second lid and the inner wall of the third lid, and the water volume in the water injection zone does not exceed the upper surface of the isolation plate on the horizontal plane.
[0021] Furthermore, the incubation time is 4 to 6 hours.
[0022] Furthermore, the fibroblasts are selected from porcine fibroblasts, bovine fibroblasts, sheep fibroblasts, porcine ear tissue fibroblasts, bovine ear tissue fibroblasts, sheep ear tissue fibroblasts, porcine kidney fibroblasts, bovine kidney fibroblasts, and sheep kidney fibroblasts.
[0023] Furthermore, the plasmid containing the target gene is selected as PEGFp-N1.
[0024] Furthermore, the plasmid solution containing the linearized target gene is obtained by purifying the plasmid containing the target gene using Ase I enzyme digestion.
[0025] Furthermore, each adherent fibroblast is injected with a plasmid solution containing the linearized target gene at a concentration of 150–200 ng / μL in a volume of 1 ± 0.2 pL.
[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0027] This invention provides a fibroblast gene transfection method that eliminates the need for further screening. Therefore, the injected gene does not require a selection marker, thus achieving the goal of obtaining a cell line without a selection marker. Furthermore, through extensive experimental exploration, this invention achieves a stable transfection efficiency of up to 92.3% by coordinating parameters such as the preparation of the injection cell suspension droplets (including a fibroblast suspension concentration of 1-2 droplets / µL, each droplet being formed from 10±0.5µL of the fibroblast suspension, and incubation of the droplets in a microenvironment), adjusting the injection needle angle (30-60°), and the target gene concentration (150-200 ng / µL). Attached Figure Description
[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0029] Figure 1 This is a schematic diagram of the microenvironment of a multi-layered dish; Figure 1 The accompanying diagram is labeled as follows: 1. 35mm cell culture dish body; 2. First dish lid; 3. Second dish lid; 31. Vent hole; 4. Third dish lid; 5. Separator; 6. 110mm cell culture dish body; 7. Water filling area;
[0030] Figure 2 Schematic diagram of cell suspension droplets; Figure 2 The accompanying diagram is labeled as follows: 8, cell suspension droplets; L, the distance from the edge of the cell culture dish to the seeding position of the droplet closest to the edge of the cell culture dish;
[0031] Figure 3 This is a schematic diagram of the injection needle installation angle; α is the angle between the injection needle and the horizontal plane;
[0032] Figure 4 This is a schematic diagram of the injection process;
[0033] Figure 5 Schematic diagram of cells 48h (left) and 16d after EGFP injection (right). Detailed Implementation
[0034] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.
[0035] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.
[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.
[0037] This invention provides a method for gene transfection of fibroblasts, the general idea of which is as follows:
[0038] Those skilled in the art will know that electroporation or liposome transfection can only introduce exogenous genes with selection markers to obtain cell lines with selection markers. Somatic cell clones of transgenic pigs obtained by somatic cell nuclear transfer technology from these selected cells also carry selection markers, making it impossible to propagate this pig breed to obtain a population.
[0039] Those skilled in the art generally do not use the microinjection method because the microinjection method has the disadvantages of limited transfection quantity, low transfection rate, and inability to achieve stable transfection.
[0040] Through extensive experimentation, the inventors of this application discovered that by adjusting parameters such as the angle of the injection needle (preferably 30–60 degrees), the cell culture microenvironment after injection, the preparation of the cell suspension droplets for injection and their cell density, and the concentration of the target gene (between 150–200 ng / uL), a stable transduction efficiency of 92.3% was ultimately achieved.
[0041] Therefore, the present invention provides a method for gene transfection of fibroblasts, the method specifically comprising:
[0042] Step S1: Obtain a fibroblast suspension with a cell concentration of 1-2 cells / µL. Prepare cell suspension droplets in a cell culture dish using the fibroblast suspension. Each cell suspension droplet is formed from 10±0.5µL of the fibroblast suspension.
[0043] As an optional implementation, the fibroblasts are selected from porcine fibroblasts, bovine fibroblasts, sheep fibroblasts, porcine ear tissue fibroblasts, and kidney fibroblasts. In other implementations, the fibroblasts can be porcine fibroblasts, bovine fibroblasts, sheep fibroblasts, or, in other implementations, porcine (bovine / sheep) ear tissue fibroblasts and porcine (bovine / sheep) kidney fibroblasts. As a specific implementation, the fibroblasts are fetal fibroblasts from Hubei White pigs.
[0044] As an optional implementation, in step S1,
[0045] The specific preparation steps for obtaining a fibroblast suspension with a cell concentration of 1-2 cells / µL include:
[0046] Step S101: Obtain a fibroblast suspension with a cell concentration of 1-2 cells / µL.
[0047] (1) Isolation of fibroblasts from Hubei White pig fetuses: Hubei White pig fetuses that had reached 35 days of development were removed from their mothers and washed 6-8 times with DPBS (Dubor's phosphate buffer) containing 1% penicillin-streptomycin antibiotics on a sterile laminar flow hood to remove as much exogenous contaminant as possible. The muscle tissue from the back and buttocks was removed with sterilized forceps and cut into pieces of approximately 1 mm in volume. 3 Tissue fragments were carefully removed, removing as much viscera and head tissue as possible to prevent contamination with other cell types. The tissue fragments were digested with trypsin and cultured in an incubator at 39°C, saturated humidity, and 5% CO2. The culture medium was DMEM supplemented with 10% fetal bovine serum.
[0048] (2) Primary culture of fetal fibroblasts from Hubei White pigs: Closely observe the cells cultured in step (1). After 3-5 days of initial culture, fibroblasts can be observed spreading from the edge of the tissue block outwards. At this time, replace the culture medium with fresh medium every 3 days or so until the fibroblasts cover the entire bottom of the culture plate.
[0049] (3) Passage culture of fetal fibroblasts from Hubei White pigs: When the confluence of primary cells in step (2) reaches 100%, discard the cell culture medium in the culture dish and wash twice with DPBS (Dubor's phosphate buffer) preheated to 39°C and containing 1% penicillin-streptomycin antibiotics to completely remove residual culture medium. Add trypsin to digest the cells, then add an equal volume of complete culture medium to neutralize the trypsin. Pipette the cells to form a single-cell suspension, divide them evenly into 2-3 culture flasks, and culture them in an incubator at 39°C, saturated humidity, and 5% CO2.
[0050] (4) Cryopreservation of fetal fibroblasts from Hubei White pigs: When the confluence of passaged cells reached 100%, discarded culture medium was removed, the cells were washed with DPBS, and digested with trypsin to form a single-cell suspension. The suspension was then transferred to freshly sterilized 1.5ml centrifuge tubes and centrifuged at 6000rpm for 2min on a benchtop centrifuge to collect the cells at the bottom of the tube. The supernatant was thoroughly removed, and cryopreservation solution was added. The cryopreservation solution was formulated as DMEM:fetal bovine serum:DMSO = 7:2:1. The cryopreservation tubes containing the cells to be cryopreserved were placed in a foam cryopreservation box and then placed together in an ultra-low temperature freezer at -80℃ overnight. After 24 hours, the cells were transferred to liquid nitrogen for long-term storage. The cryopreservation date, sample name, and operator should be clearly labeled.
[0051] (5) Resuscitation of fetal fibroblasts from Hubei White pigs: One tube of frozen cells was taken out from liquid nitrogen and quickly placed in a 37°C water bath with constant shaking to thaw them. After aspirating the cells, they were added to a 1.5mL centrifuge tube, centrifuged at 3000rpm for 2min30s, the supernatant was discarded, 1mL of cell culture medium was added and gently pipetted to mix well. The cell suspension was then added to a cell culture dish, and an appropriate volume of complete culture medium was added. The mixture was gently shaken to mix well and cultured in an incubator at 39°C, saturated humidity, and 5% CO2.
[0052] (6) Obtaining a fibroblast suspension:
[0053] When the recovered cells reach approximately 80% confluence, digest them with trypsin (0.25% concentration) diluted 1:1 with DPBS. When cell edges begin to shrink under a microscope, add 1 mL of culture medium and pipette, collecting the cells into a 1.5 mL EP tube. Incubate at 2500 rpm for 2 minutes at room temperature, discarding the supernatant. Dilute the cell pellet with DMEM in 10% FBS at a ratio of 1:1000 and count the cells under a microscope. The cell concentration should be 1–2 cells / µL (preferably, 1 cell per µL of culture medium is optimal).
[0054] Step S102: Prepare cell suspension microdroplets by placing the fibroblast suspension in a cell culture dish.
[0055] The cell suspension droplets are prepared in cell culture dishes with a diameter of 35 mm, with 6 ± 1 droplets prepared in each 35 mm diameter cell culture dish. Each cell suspension droplet is formed from 10 ± 0.5 μL of the fibroblast suspension; preferably, each droplet is 10 μL, and it is preferable to prepare 6 droplets in each cell culture dish. Preferably, during inoculation, the straight-line distance L between the inoculation position of the droplet closest to the edge of the cell culture dish and the edge of the cell culture dish (on the diameter of the cell culture dish) is L = 1 ± 0.2 cm (e.g., ...). Figure 2 As shown in the figure, the inventors of this application have discovered that if the inoculation site is too close to the edge of the cell culture dish, it is not conducive to subsequent gene injection operations and the cell survival rate is low. Figure 2 Preferred arrangement and position of 6 cell suspension droplets
[0056] The specific operation of preparing cell suspension droplets from the fibroblast suspension in a cell culture dish can be performed using a pipette with a volume range of 2-20 μL.
[0057] Step S2: Incubate the cell culture dish containing cell suspension droplets in a static environment. After the cells adhere to the dish, add them to a cell culture incubator for further culture.
[0058] After cell adhesion, each cell suspension droplet formed a cell cluster consisting of approximately 10 fibroblasts. Six cell suspension droplets (e.g.) Figure 2 This means there are 6 cell groups, in which cells are relatively concentrated. This is because the more concentrated the cells are, the easier it is for them to reproduce.
[0059] In a preferred embodiment, a cell culture dish containing cell suspension droplets is placed in a sleeved microenvironment for incubation. This sleeved microenvironment is formed by combining multiple cell culture dishes, such as... Figure 1 As shown, it specifically includes:
[0060] The cell culture dish containing cell suspension droplets is a 35mm diameter cell culture dish, comprising a 35mm diameter dish body and a first dish lid;
[0061] The first dish cover is covered by the second dish cover, which is the cover of a cell culture dish with a diameter of 90 mm;
[0062] The second dish cover is covered by a third dish cover, which is the cover of a cell culture dish with a diameter of 110 mm;
[0063] The bottom of the 35mm diameter dish is provided with a partition plate, and the lower end of the second dish lid is located on the partition plate;
[0064] The bottom of the isolation plate is provided with a cell culture dish with a diameter of 110 mm, and the lower end of the third dish cover is located on the cell culture dish with a diameter of 110 mm.
[0065] The second lid has one or more vent holes at its top; a water injection zone is formed between the outer wall of the second lid and the inner wall of the third lid, and the water volume in the water injection zone does not exceed the upper surface of the isolation plate on the horizontal plane.
[0066] The reason for placing the cell culture dish containing cell suspension droplets in the microenvironment of the sleeve dish is as follows: (1) The microenvironment formed by the sleeve dish can effectively reduce the changes in the liquid and gaseous environments caused by external factors, and can ensure that the environment in which the cells (in the 35mm culture dish) are located remains stable, thereby improving cell survival rate and reproduction efficiency. (2) Figure 2 All culture dishes used were sterile, and the water used in the liquid phase environment was autoclaved to ensure that the microenvironment would not contaminate the cells. (3) A ventilation hole 31 was properly set at the top of the 90mm culture dish with a red-hot needle to ensure the circulation of gas between the internal and external environments. (4) Figure 2 The middle isolation plate 5 is sterilized and used to place 35mm culture dishes. The sterile water level in the water injection area must not exceed the height of the isolation plate to prevent water from the water injection area from entering the 35mm culture dish containing the cells.
[0067] In step S2, the incubation time is 4–6 hours. Incubation in the microenvironment of the dish for 4–6 hours is beneficial for cell adhesion and high cell survival rate. After cell adhesion, 2.5 mL of DMEM cell culture medium containing 10% FBS is added and the cells are cultured in an incubator at 39°C, saturated humidity, and 5% CO2 for later use.
[0068] Step S3: Obtain a plasmid solution containing the linearized target gene at a concentration of 150-200 ng / uL.
[0069] The inventors of this application have discovered that if the concentration of the plasmid solution containing the linearized target gene is less than 150 ng / uL, it will result in very low gene expression.
[0070] Interestingly, the inventors of this application found that if the concentration of the plasmid solution containing the linearized target gene was greater than 200 ng / uL, many cells died. The specific reason is unknown, but it is speculated that it may be due to the high osmotic pressure.
[0071] The plasmid containing the target gene can be any plasmid containing the target gene of interest;
[0072] As an optional implementation method, the target gene used is pEGFP-N1, which was purchased from Zhili Zhongte Biotechnology, platform number: zl-059451.
[0073] As an optional implementation, the plasmid solution containing the linearized target gene is obtained by purifying the plasmid containing the target gene using AseI enzyme digestion. In other implementations, other enzyme digestions may be used for linearization.
[0074] Step S4: Place the culture dish containing cells on the stage, draw the plasmid solution containing the linearized target gene into the injection needle, adjust the angle between the injection needle and the horizontal plane to 30-60°, and inject into each cell in sequence; after injection, perform single-cell culture to obtain stably transfected transgenic cell clones.
[0075] In step S4
[0076] The reason for adjusting the angle between the injection needle and the horizontal plane to 30-60° in this application is that if the angle is too large, it is easy to cause the needle tip to break, requiring frequent needle changes and prolonged exposure of cells, which makes cell survival difficult; if the angle is too small, the needle tip may easily lift the cells, causing them to die.
[0077] Each cell suspension droplet adhered to the culture medium, resulting in 10 adherent fibroblasts. Each fibroblast was injected with a plasmid solution containing the linearized target gene at a concentration of 150–200 ng / μL, in a volume of 1 ± 0.2 pL (1 μL = 10 μg / μL). 6pL), preferably 1 pL.
[0078] As a specific implementation method: Place the injection needle in linearized DNA of a pre-adjusted concentration, and use the siphon principle to draw in the target gene to be injected. Then connect the injection needle to a micro-automatic injection device. After completing the operation, center the needle tip in the field of view of the objective lens. It is crucial to operate under a microscope at this stage to prevent breaking the needle tip. After preparation, place the culture dish containing cells on the stage and adjust the injection needle to 30–60°.
[0079] The following will provide a detailed description of a fibroblast gene transfection method of this application, in conjunction with embodiments and experimental data.
[0080] Example 1: A method for gene transfection of primary porcine fetal fibroblasts
[0081] 1. Obtaining fibroblasts from fetal Hubei white pigs
[0082] (1) Isolation of fibroblasts from Hubei White pig fetuses: Hubei White pig fetuses that had reached 35 days of development were removed from their mothers and washed 6-8 times with DPBS (Dubor's phosphate buffer) containing 1% penicillin-streptomycin antibiotics on a sterile laminar flow hood to remove as much exogenous contaminant as possible. The muscle tissue from the back and buttocks was removed with sterilized forceps and cut into pieces of approximately 1 mm in volume. 3 Tissue fragments were carefully removed, removing as much viscera and head tissue as possible to prevent contamination with other cell types. The tissue fragments were digested with trypsin and cultured in an incubator at 39°C, saturated humidity, and 5% CO2. The culture medium was DMEM supplemented with 10% fetal bovine serum.
[0083] (2) Primary culture of fetal fibroblasts from Hubei White pigs: Closely observe the cells cultured in step (1). After 3-5 days of initial culture, fibroblasts can be observed spreading from the edge of the tissue block outwards. At this time, replace the culture medium with fresh medium every 3 days or so until the fibroblasts cover the entire bottom of the culture plate.
[0084] (3) Passage culture of fetal fibroblasts from Hubei White pigs: When the confluence of primary cells in step (2) reaches 100%, discard the cell culture medium in the culture dish and wash twice with DPBS (Dubor's phosphate buffer) preheated to 39°C and containing 1% penicillin-streptomycin antibiotics to completely remove residual culture medium. Add trypsin to digest the cells, then add an equal volume of complete culture medium to neutralize the trypsin. Pipette the cells to form a single-cell suspension, divide them evenly into 2-3 culture flasks, and culture them in an incubator at 39°C, saturated humidity, and 5% CO2.
[0085] (4) Cryopreservation of fetal fibroblasts from Hubei White pigs: When the confluence of passaged cells reached 100%, discarded culture medium was removed, the cells were washed with DPBS, and digested with trypsin to form a single-cell suspension. The suspension was then transferred to freshly sterilized 1.5ml centrifuge tubes and centrifuged at 6000rpm for 2min to collect the cells at the bottom of the tube. The supernatant was thoroughly removed, and cryopreservation solution was added. The cryopreservation solution was formulated as DMEM:fetal bovine serum:DMSO = 7:2:1. The cell cryopreservation tubes containing the cells were placed in a foam cryopreservation box and then placed together in a -80℃ ultra-low temperature freezer overnight. After 24 hours, the cells were transferred to liquid nitrogen for long-term storage. The cryopreservation date, sample name, and operator should be clearly labeled.
[0086] (5) Resuscitation of fetal fibroblasts from Hubei White pigs: One tube of frozen cells was taken out from liquid nitrogen and quickly placed in a 37°C water bath with constant shaking to thaw them. The cells were aspirated and added to a 1.5mL centrifuge tube, centrifuged at 3000rpm for 2min30s, the supernatant was discarded, 1mL of cell culture medium was added and gently pipetted to mix well, the cell suspension was added to a cell culture dish, and an appropriate volume of complete culture medium was added. The mixture was gently shaken to mix well and cultured in an incubator at 39°C, saturated humidity, and 5% CO2.
[0087] (6) Preparation of microdroplets of fetal fibroblast suspension from Hubei White pigs:
[0088] When the recovered cells reached approximately 80% confluence, trypsin was diluted 1:1 with DPBS and the cells were digested. When cell shrinkage was observed under a microscope, 1 mL of culture medium was added and the mixture was thoroughly mixed. The cells were collected into 1.5 mL EP tubes and incubated at 2500 rpm for 2 minutes at room temperature. The supernatant was discarded. The cell pellet was diluted 1:1000 with DMEM containing 10% FBS, and the cells were counted under a microscope. The optimal concentration was 1 cell per μL of culture medium. Cell suspension droplets were prepared in 35 mm disposable cell culture dishes, 1 cm from the edge of the dish. Each droplet contained 10 μL, and 6 droplets were prepared in each cell culture dish (see attached diagram). Figure 2 Cell dishes containing cell suspension droplets were placed in a microenvironment and incubated for 4–6 hours. After cell adhesion, 2.5 mL of DMEM cell culture medium containing 10% FBS was added and cultured in an incubator at 39°C, saturated humidity, and 5% CO2 for later use.
[0089] The microenvironment of the cell culture dish is formed by combining multiple cell culture dishes, specifically including:
[0090] The cell culture dish containing cell suspension droplets is a 35mm diameter cell culture dish, comprising a 35mm diameter dish body and a first dish lid;
[0091] The first dish cover is covered by the second dish cover, which is the cover of a cell culture dish with a diameter of 90 mm;
[0092] The second dish cover is covered by a third dish cover, which is the cover of a cell culture dish with a diameter of 110 mm;
[0093] The bottom of the 35mm diameter dish is provided with a partition plate, and the lower end of the second dish lid is located on the partition plate;
[0094] The bottom of the isolation plate is provided with a cell culture dish with a diameter of 110 mm, and the lower end of the third dish cover is located on the cell culture dish with a diameter of 110 mm.
[0095] The second lid has one or more vent holes at its top; a water injection zone is formed between the outer wall of the second lid and the inner wall of the third lid, and the water volume in the water injection zone does not exceed the upper surface of the isolation plate on the horizontal plane.
[0096] 2. Preparation of target gene for injection
[0097] (1) Propagation of target gene
[0098] The target gene pEGFP-N1 used in this experiment was purchased from Zhili Zhongte Biotechnology, platform number: zl-059451.
[0099] Mix 10 μL of the target gene solution with 100 μL of competent cells, spread evenly on an LB solid culture plate containing kanamycin, invert, and incubate overnight at 37°C. Observe the growth of colonies the next morning, and select colonies for propagation in LB liquid medium for plasmid extraction.
[0100] (2) Plasmid extraction
[0101] First, prepare LB liquid medium and add kanamycin resistance. Then, inoculate 40 μL of pEGFP-N1 bacterial culture into 100 mL of LB liquid medium (containing kanamycin), and incubate overnight at 37°C. The next morning, extract the plasmid using Tiangen's mini plasmid extraction kit (see kit instructions for specific steps). The concentration and OD value of the extracted plasmid were measured using a spectrophotometer and verified by gel electrophoresis.
[0102] (3) Plasmid linearization: The enzyme digestion reaction system is shown in Table 1:
[0103] Table 1
[0104]
[0105] After mixing, centrifuge briefly and incubate at 37°C for 2–4 hours. Electrophoresis is used to verify the digestion effect after the enzyme digestion is complete.
[0106] (4) Purification of linearized plasmids
[0107] The enzyme-digested product was extracted once with an equal volume of chloroform (retaining the supernatant), and this step was repeated. 1 / 10 volume of 3 mol / L sodium acetate (pH 5.0–5.2) was added, followed by 2 volumes of pre-chilled anhydrous ethanol. The mixture was incubated at -20°C for 30 min, centrifuged at 12000 rpm for 10 min, and the precipitate was washed once with 75% ethanol. The mixture was then centrifuged again at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was allowed to air dry. The precipitate was dissolved in 20–40 μL of TE buffer (10 mM Tris, 0.15 mM EDTA, pH 7.2–7.4). The concentration and OD value were measured using a spectrophotometer, and the final concentration was adjusted to 200 ng / μL.
[0108] 3. Intracellular gene injection
[0109] Cell preparation: Replace the culture medium with fresh one for the cells to be injected to prevent cell debris in the old culture medium from clogging the injection needle. Observe under a microscope to ensure that the cell culture medium is clear and clean.
[0110] Preparation of the injection needle: Place the refractive needle into the linearized DNA solution of the pre-adjusted concentration. Use the siphon principle to draw in the target gene to be injected. Connect the injection needle to the automated microinjector. After the operation is complete, center the needle tip in the field of view of the objective lens. This process must be performed under a microscope to prevent breakage of the needle tip. After preparation, place the culture dish containing cells on the stage and adjust the injection needle to a convenient 45-degree angle for injection. Each microdroplet should inject 1 pL of the linearized plasmid.
[0111] Each culture dish contains 6 microdroplets, and each microdroplet contains 10 cells; each experiment sets up 3 flat control groups, for a total of 18 microdroplets and 180 cells.
[0112] 4. Obtaining transgenic cells
[0113] After injection, the cells are placed in a dome-shaped microenvironment for culture; see appendix for details. Figure 1 The culture medium was replaced with fresh medium every 24 hours, and half of the medium was replaced every 48 hours, until cell growth significantly improved. Then the entire culture medium was replaced, and the cells were cultured for subsequent experiments. A stable transformation efficiency of 92.3% was ultimately achieved. See attached diagram for details. Figure 4 .
[0114] Example 2: A method for gene transfection of primary porcine fetal fibroblasts
[0115] The method for gene transfection of porcine primary fetal fibroblasts provided in Embodiment 2 of the present invention is the same as in Embodiment 1, except that the angle between the injection needle and the horizontal plane is changed to 30°.
[0116] Example 3: A method for gene transfection of primary porcine fetal fibroblasts
[0117] The method for gene transfection of porcine primary fetal fibroblasts provided in Embodiment 3 of the present invention is the same as in Embodiment 1, except that the angle between the injection needle and the horizontal plane is changed to 60°.
[0118] Example 4: A method for gene transfection of primary porcine fetal fibroblasts
[0119] The present invention provides a method for gene transfection of porcine primary fetal fibroblasts, except that the concentration of the plasmid solution containing the linearized target gene is 150 ng / uL, and the other operations are the same as in Example 1.
[0120] Example 5: A method for gene transfection of primary porcine fetal fibroblasts
[0121] The method for gene transfection of porcine primary fetal fibroblasts provided in Example 5 of this invention is the same as in Example 1, except that the concentration of the plasmid solution containing the linearized target gene is 130 ng / uL.
[0122] Comparative Example 1
[0123] In Comparative Example 1, no nested microenvironment was set up, but other operations were the same as in Example 1.
[0124] Comparative Example 2
[0125] In Comparative Example 2, the only difference was that the concentration of the plasmid solution containing the linearized target gene was adjusted to 100 ng / uL; all other operations were the same as in Example 1.
[0126] Comparative Example 3
[0127] In Comparative Example 3, the only difference was that the angle between the injection needle and the horizontal plane was changed to 70°; all other operations were the same as in Example 1. In this comparative example, the injection needle was difficult to position within the cell nucleus, resulting in unsuccessful gene transfection.
[0128] Experimental Example 1
[0129] The cell viability and transfection rate of each embodiment and comparative example are statistically analyzed, as shown in Table 2.
[0130] Table 2
[0131]
[0132]
[0133] As can be seen from Table 2,
[0134] In Comparative Example 1, without setting up a microenvironment, the cell survival rate was only 33.3%;
[0135] In Comparative Example 2, the concentration of the plasmid solution containing the linearized target gene was 100 ng / uL, which is less than the range of 150-200 ng / uL in Example 1 of the present invention, and the transfection rate was only 69.6%.
[0136] In Comparative Example 3, gene transfection failed because the angle between the injection needle and the horizontal plane was 70°, which is greater than the range of 30-60° in Example 3 of the present invention.
[0137] In Examples 1-5 of this invention, the cell viability reached 65% to 87.8%, and the transfection rate reached 88.6% to 92.3%; stable cell transfection was achieved, and cell lines without selection markers were obtained.
[0138] In summary, this invention achieves a stable transfection efficiency of up to 92.3% by coordinating parameters such as the preparation of injection cell suspension droplets (including a fibroblast suspension concentration of 1-2 droplets / µL, each droplet being formed from 10±0.5µL of the fibroblast suspension, and incubation of the droplets in a microenvironment), adjusting the injection needle angle (30-60°), and the target gene concentration (150-200ng / µL). Furthermore, the method provided by this invention directly injects genes into the cells to be transfected using a micromanipulation system, eliminating the need for further screening. Therefore, the injected gene does not require a selection marker, thus achieving the goal of obtaining a cell line without selection markers.
[0139] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0140] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.
[0141] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A method for gene transfection in fibroblasts, characterized in that, The method includes: A fibroblast suspension with a cell concentration of 1-2 cells / μL was obtained. The fibroblast suspension was seeded into cell culture dishes with a diameter of 35 mm in the form of 10±0.5 μL microdroplets, with 6±1 microdroplets seeded into each culture dish, and the outermost microdroplet was 1±0.2 cm away from the edge of the culture dish. The cell culture dish containing cell suspension droplets was incubated in a sleeved microenvironment. After cell adhesion, multiple adherent fibroblasts were formed, which were then added to a cell culture incubator for further culture. The sleeved microenvironment was formed by combining multiple cell culture dishes, specifically including: The cell culture dish containing cell suspension droplets is a 35mm diameter cell culture dish, comprising a 35mm diameter dish body and a first dish lid; The first dish cover is covered by the second dish cover, which is the cover of a cell culture dish with a diameter of 90 mm; The second dish cover is covered by a third dish cover, which is the cover of a cell culture dish with a diameter of 110 mm; The bottom of the 35mm diameter dish is provided with a partition plate, and the lower end of the second dish lid is located on the partition plate; The bottom of the isolation plate is provided with a cell culture dish with a diameter of 110 mm, and the lower end of the third dish cover is located on the cell culture dish with a diameter of 110 mm. The second dish lid has one or more ventilation holes at its top; a water injection zone is formed between the outer wall of the second dish lid and the inner wall of the third dish lid, and the water injection volume in the water injection zone does not exceed the upper surface of the isolation plate on the horizontal plane, in order to maintain the stability of the microenvironment, and the incubation time is 4 to 6 hours. After cell adhesion, multiple adherent fibroblasts are formed and added to a cell culture incubator for culture. A plasmid solution containing the linearized target gene with a concentration of 150-200 ng / μL is obtained. The plasmid solution is drawn into an injection needle, and the angle between the injection needle and the horizontal plane is adjusted to 30-60°. 1±0.2 pL of the plasmid solution is injected into each adherent fibroblast. Single-cell culture was performed after injection to obtain transgenic cell clones that were stably transfected without selection markers.
2. The method for gene transfection of fibroblasts according to claim 1, characterized in that, After cell adhesion, add 2.5 mL of DMEM cell culture medium containing 10% FBS and culture in an incubator at 39°C, saturated humidity, and 5% CO2 for later use.
3. The method for gene transfection of fibroblasts according to claim 1, characterized in that, The fibroblasts are selected from porcine fibroblasts, bovine fibroblasts, sheep fibroblasts, porcine ear tissue fibroblasts, bovine ear tissue fibroblasts, sheep ear tissue fibroblasts, porcine kidney fibroblasts, bovine kidney fibroblasts, and sheep kidney fibroblasts.
4. The method for gene transfection of fibroblasts according to claim 1, characterized in that, The cell concentration of the fibroblast suspension was 1 cell / µL.
5. The method for gene transfection of fibroblasts according to claim 1, characterized in that, The plasmid containing the target gene is pEGFP-N1.
6. The method for gene transfection of fibroblasts according to claim 1, characterized in that, The plasmid solution containing the linearized target gene was obtained by purifying the plasmid containing the target gene using AseI enzyme digestion.