Method for constructing mitochondrial DNA depleted Rho0 cell line
The mitochondrial DNA-depleted Rho0 cell line was constructed by treating ethidium bromide-tolerated cell line by tacitabine, solving the problems of safety and efficiency in the prior art, and achieving low toxicity and efficient cell line construction.
Patent Information
- Application Number
- CN202510469794.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to safely and reliably construct Rho0 cell lines depleted with mitochondrial DNA, and traditional drugs such as ethidium bromide are carcinogenic, which limits their practical application.
The ethidium bromide-tolerated cell lines were treated with taxitabine, and the Rho0 cell lines with mitochondrial DNA were continuously cultured through culture medium containing taxitabine.
A safe and low toxic mitochondrial DNA depletion effect is achieved, shortening cell line construction time and reducing costs and improving construction efficiency.
Smart Images
Figure CN120290481A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, and particularly to a method for constructing a Rho0 cell line with mitochondrial DNA depletion. Background Art
[0002] Mitochondrial DNA (mtDNA) plays a central role in mitochondrial biology research and is almost involved in and affects all cellular processes. Knocking out genes required for mtDNA replication in animals is usually embryonically lethal, while knocking out the same type of genes in cells cultured in vitro can achieve mtDNA depletion, thereby obtaining a Rho-0 (ρ0) cell line lacking mtDNA.
[0003] Currently, there are some technical methods to obtain ρ0 cells, including treating cells with mtDNA replication inhibitors such as ethidium bromide (EtBr, EB), xylene, etc. However, due to different cell characteristics, some cell lines can quickly recover their mtDNA copy number, while some cell lines are intolerant and die quickly, and cannot be cultivated into ρ0 cells by this method. Moreover, these two drugs are carcinogenic, which also greatly limits their practical applications.
[0004] Therefore, how to safely and reliably construct a cell line with mitochondrial DNA depletion is a problem to be solved. Summary of the Invention
[0005] The embodiments of this application provide a method for constructing a Rho0 cell line with mitochondrial DNA depletion to solve the problems existing in the related technologies. The technical solutions are as follows:
[0006] In a first aspect, the embodiments of this application provide a method for constructing a Rho0 cell line with mitochondrial DNA depletion, including the following steps:
[0007] (1) Cultivate cells using a culture medium containing ethidium bromide, and screen for tolerant and insensitive cell lines;
[0008] (2) Cultivate the cell lines;
[0009] (3) Treat the cell culture medium with a drug containing zalcitabine and continue to cultivate to obtain a Rho0 cell line with mitochondrial DNA depletion.
[0010] In an implementation manner, in step (1), when cultivating cells using a culture medium containing ethidium bromide, the concentration of ethidium bromide in the culture medium is 20 - 40 ng / ml.
[0011] In one embodiment, the culture medium is DMEM medium containing 10% FBS, 1% P / S double antibiotics, 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine.
[0012] In one embodiment, cell culture is carried out in an incubator at 37°C and 5% CO2.
[0013] In one embodiment, in step (2), after the cell line is revived, it is passaged 2 times and cultured to adhere and grow for 10 - 12 h.
[0014] In one embodiment, the cells are placed in DMEM medium containing 10% FBS and 1% P / S double antibiotics; the culture is carried out in an incubator at 37°C and 5% CO2.
[0015] In one embodiment, when the cell culture medium is treated with a drug containing zalcitabine and continued to be cultured in step (3),
[0016] The drug-treated cell culture medium is DMEM medium containing 10% FBS, 1% P / S double antibiotics, 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine.
[0017] In one embodiment, the concentration of zalcitabine in the drug-treated cell culture medium is 5 - 10 μM;
[0018] In one embodiment, the time for continued culture is 7 - 22 days.
[0019] In one embodiment, when the cell culture medium is treated with a drug containing zalcitabine and continued to be cultured in step (3), the culture is carried out at a high zalcitabine concentration and a low zalcitabine concentration in sequence.
[0020] In one embodiment, the high zalcitabine concentration is 5 - 10 μM; the low zalcitabine concentration is 40 - 60% of the high zalcitabine concentration.
[0021] In one embodiment, the culture is carried out for 7 - 15 days at the high zalcitabine concentration; the culture is carried out for 5 - 7 days at the low zalcitabine concentration.
[0022] In one embodiment, when continuing to culture in the drug-treated cell culture medium at the high zalcitabine concentration, the culture medium is changed daily.
[0023] In one embodiment, after continuing to culture the cell culture medium with a drug containing zalcitabine, it further includes a cell cloning and cell culture amplification process to obtain the Rho0 cell line with depleted mitochondrial DNA.
[0024] In one embodiment, when the cell culture medium is treated with a drug containing zalcitabine and continued to be cultured, ethidium bromide is added to the culture medium for culturing at the same time.
[0025] In one embodiment, the concentration of ethidium bromide in the culture medium is 10 - 30 ng / ml.
[0026] In one embodiment, the cell is A375 cell.
[0027] The advantages or beneficial effects in the above technical solutions at least include:
[0028] This application uses zalcitabine to treat ethidium bromide-resistant cells to obtain a Rho0 cell line with depleted mitochondrial DNA; the reagents, consumables, operation methods and techniques adopted are safe and have low toxicity; it has more obvious effects and higher efficiency, can shorten the time for cell line construction and reduce costs.
[0029] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of this application will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In the drawings, unless otherwise specified, the same reference numerals throughout the several views refer to the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed according to this application and should not be regarded as limiting the scope of this application.
[0031] Figure 1 Shows the depletion of mitochondrial DNA in cells after 7 days of treatment with the inhibitor;
[0032] Figure 2 Shows the depletion of mitochondrial DNA in cells after 15 days of treatment with the inhibitor. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0033] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of this application. Therefore, the drawings and the description are considered to be exemplary in nature and not restrictive.
[0034] Zalcitabine (ddC) is an analogue of the cellular DNA precursor dCTP and was initially developed as an antiretroviral drug against HIV infection. However, it has side effects such as peripheral neuropathy, stomatitis, and oral ulcers, which have been confirmed to be related to the inhibition of mitochondrial DNA polymerase γ (POLG). Taking the EB-tolerant cancer cell A375 as an example, we conducted this study on whether ddC can induce mtDNA depletion in cultured cells and its induction efficiency. The specific methods are as follows:
[0035] I. Cell line screening
[0036] When culturing cells in vitro using DMEM medium containing 10% FBS (fetal bovine serum), 1% P / S double antibiotics (penicillin / streptomycin), 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine in a 37°C, 5% CO2 incubator, ethidium bromide was added to make the concentration of ethidium bromide 20 - 40 ng / ml; and continuous passage was carried out to observe and screen the EB-treated tolerant and insensitive cell line A375.
[0037] II. Experimental procedures.
[0038] Cell culture medium: DMEM medium, containing 10% FBS, 1% P / S double antibiotics;
[0039] Drug-treated cell culture medium: On the basis of the above cell culture medium, 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine were added;
[0040] The cells were placed in a 37°C, 5% CO2 incubator.
[0041] The specific experimental procedures are as follows:
[0042] S1. Take out the preserved A375 cell line from the liquid nitrogen tank for culture. After resuscitating and passing the cells 2 times, seed the cells in a 6-well plate at a density of 1*10 5 cells / ml;
[0043] S2. After the cells adhered and grew for 12 h, add drugs to start treating the cells; The experimental groups were set as Control group, L-E group (EB 20 ng / ml EB), H-E group (EB 40 ng / ml), L-d group (ddC 5 μM), H-d group (ddC 10 μM), and E+d group (EB20 ng / ml, ddC 5 μM);
[0044] S3. Thereafter, all the cells were passaged once every two days, successively from 6-well plates to 5-cm culture dishes and then to 10-cm culture dishes, and were continuously treated with drugs. The six groups of cells were set as follows: Control group, L-E group (EB 20 ng / ml EB), H-E group (EB 40 ng / ml), L-d group (ddC 5 μM), H-d group (ddC 10 μM), and E+d group (EB 20 ng / ml, ddC 5 μM);
[0045] S4. After being passaged to 10-cm culture dishes, they were subcultured in 10-cm culture dishes at a ratio of 1:2, and the drug treatment concentration remained unchanged;
[0046] S5. After 7 days of continuous drug treatment, some cells were taken, and DNA was extracted using the StarSpin Column Universal DNA Extraction Kit from Kangrun Biotech; 10 ng of DNA was taken for fluorescence quantitative PCR experiment (qPCR) to detect the change in mitochondrial DNA copy number; the detected fragments included: COX1, COX2, Dloop, ND6, 36B4 (nuclear reference gene); the results of the stage-by-stage detection of mtDNA copy number were as Figure 1 shown;
[0047] S6. Since the reduction of mtDNA copy number leads to the decrease of mitochondrial oxidative phosphorylation function and the cells will produce a large amount of lactic acid, the culture medium was changed every day; at this stage, the cells were subcultured at a ratio of 1:2, and some cells were taken after 8 days to extract DNA for qPCR to detect the mitochondrial DNA copy number; the detected fragments included: COX1, COX2, Dloop, ND6, 36B4 (nuclear reference gene); the results of the stage-by-stage detection of mtDNA copy number were as Figure 2 shown;
[0048] S7. The drug treatment concentration of each group was reduced, and the changed concentration was as follows: L-E group 10 ng / ml EB, H-E group 20 ng / ml EB, L-d group 2.5 μM ddC, H-d group 5 μM ddC, E+d group 10 ng / ml EB, 2.5 μM ddC; this stage of treatment lasted for 7 days, and the mtDNA copy number was detected;
[0049] S8. Cloning was carried out by the limiting dilution method, and monoclonal cells were selected for culture and amplification; the cells obtained after trypsin digestion were suspended in the drug-treated cell culture medium, the cell density was adjusted to 10 cells / ml, 100 μl was taken and seeded in a 96-well cell culture plate, and after 7 days of growth, when positive clone wells were observed, the cells were successively passaged to 24-well plates and 6-well plates for expansion culture; the Rho0 cell line with depleted mitochondrial DNA was obtained.
[0050] From Figure 1It can be seen that after 7 days of inhibitor treatment, the mitochondrial DNA copy numbers of 6 groups of cells were detected; the results showed that the mtDNA copy number in the ddC group was significantly lower than that in the EB group, and the efficiency of ddC-induced mtDNA depletion was significantly higher than that of EB.
[0051] From Figure 2 It can be seen that after 22 days of inhibitor treatment, the mitochondrial DNA copy numbers of 6 groups of cells were detected; the results showed that the mtDNA copy number in the ddC group was significantly lower than that in the EB group, and the efficiency of ddC-induced mtDNA depletion was significantly higher than that of EB; among them, the H-d group (treated with 10 μM ddC for 15 days and 5 μM ddC for 7 days) was the optimal experimental condition for inducing and maintaining the ρ0 cell state.
[0052] In summary, when constructing a Rho0 cell line with mitochondrial DNA depletion, for cells resistant to EB in this application, zalcitabine was selected as the inhibitor for treatment, and the mitochondrial DNA copy number could be reduced to 0, obtaining a Rho0 cell line with mitochondrial DNA depletion. The reagents, consumables, operation methods and techniques used are safe and have low toxicity; compared with EB, it has more obvious effects and higher efficiency, can shorten the time for cell line construction and reduce costs.
[0053] In the description of this specification, the descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.
[0054] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" can explicitly or implicitly include at least one of such features. In the description of the present application, "a plurality" means two or more, unless otherwise specifically defined.
[0055] The above is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed in the present application, and these should all be covered by the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims
1. A method for constructing a Rho0 cell line with mitochondrial DNA depletion, characterized in that, It includes the following steps: (1) Cultivate cells in a medium containing ethidium bromide, and screen for tolerant and insensitive cell lines; (2) Culture the cell lines; (3) Treat the cell medium with a drug containing zalcitabine and continue culturing to obtain a Rho0 cell line with depleted mitochondrial DNA.
2. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein in step (1), cells are cultured in a medium containing ethidium bromide, and the concentration of ethidium bromide in the medium is 20 - 40 ng / ml; the medium is DMEM medium containing 10% FBS, 1% P / S double antibiotics, 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine; Cell culture is carried out in an incubator at 37°C and 5% CO2.
3. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein in step (2), after the cell lines are resuscitated, they are passaged 2 times and cultured to adhere and grow for 10 - 12 h; The cells are placed in DMEM medium containing 10% FBS and 1% P / S double antibiotics; culture is carried out in an incubator at 37°C and 5% CO2.
4. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein when step (3) treats the cell medium with a drug containing zalcitabine and continues culturing, the drug-treated cell medium is DMEM medium containing 10% FBS, 1% P / S double antibiotics, 50 mg / L uracil, 100 mg / L sodium pyruvate, and 2 mM glutamine; the concentration of zalcitabine in the drug-treated cell medium is 5 - 10 μM; the time for continuous culturing is 7 - 22 days.
5. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein when step (3) treats the cell medium with a drug containing zalcitabine and continues culturing, it is carried out at high and low zalcitabine concentrations in sequence; the high zalcitabine concentration is 5 - 10 μM; the low zalcitabine concentration is 40 - 60% of the high zalcitabine concentration.
6. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 5, wherein culturing at the high zalcitabine concentration lasts for 7 - 15 days; culturing at the low zalcitabine concentration lasts for 5 - 7 days.
7. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein when continuing to culture in the drug-treated cell medium with a high zalcitabine concentration, the medium is changed daily.
8. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein after treating the cell medium with a drug containing zalcitabine and continuing culturing, it further includes a cell cloning and cell culture amplification process to obtain the Rho0 cell line with depleted mitochondrial DNA.
9. The method for constructing a Rho0 cell line with depleted mitochondrial DNA according to claim 1, wherein When the cells are cultured in the cell culture medium treated with the drug containing zalcitabine in step (3), ethidium bromide is added to the culture medium for culturing at the same time.
10. The method for constructing a Rho0 cell line with mitochondrial DNA depletion according to claim 9, characterized in that the concentration of ethidium bromide in the culture medium is 10 - 30 ng / ml.