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Monoclonal cell strain of human embryo kidney cell 293T-C10 as well as preparation and application of monoclonal cell strain

A human embryonic kidney cell, monoclonal technology, applied in the biological field, can solve the problems such as the inability of virus packaging in cells, low poisoning efficiency, bacterial and mycoplasma contamination, etc., achieving low cytotoxicity, stable and reliable poisoning efficiency, obvious effect

Inactive Publication Date: 2013-02-06
上海溥生生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] We analyzed the reasons why 293T cells cannot produce virus efficiently: 1) There are potential threats of bacterial and mycoplasma contamination in 293T cells during long-term culture and passage in the laboratory, so that the cells cannot perform virus packaging in an optimal state
2) Due to the heterogeneity of 293T cells, there are both clones with high and low export efficiency in the cells, so it is possible to select 293T cells with high export efficiency from the original 293T cells through monoclonal screening clone

Method used

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  • Monoclonal cell strain of human embryo kidney cell 293T-C10 as well as preparation and application of monoclonal cell strain
  • Monoclonal cell strain of human embryo kidney cell 293T-C10 as well as preparation and application of monoclonal cell strain
  • Monoclonal cell strain of human embryo kidney cell 293T-C10 as well as preparation and application of monoclonal cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Mycoplasma was removed from 293T cells, and the state of cells before and after removal was compared.

[0048] 1. Resuscitated human embryonic kidney cell 293T cells (Institute of Cells, Chinese Academy of Sciences);

[0049] 2. Digest the recovered cells and count 1x10 5 / ml, cultivated in a 6cm dish;

[0050] 3. After adhering to the wall, replace the fresh medium, add mycoplasma removal drugs, such as quinolone derivatives MRA (MP Biomedicals 3050044), stop the drug after 1 week of culture.

[0051] 4. Take pictures to compare the morphology of cells before and after removing mycoplasma, the results are as follows figure 1 As shown, the surface of the cells after removal of mycoplasma is smooth, the cells are full, the refraction is good, and the cell growth is in good condition.

Embodiment 2

[0052] Example 2: 293T cells without mycoplasma contamination were serially diluted to culture in 96-well plates, screening and expansion experiments

[0053] 1. Digest the cells and resuspend the cells obtained in Example 1 with fresh medium.

[0054] 2. After counting, gradually dilute to 1cell / 100μl, add to 96-well plate, 37 degrees, 5% CO 2 to cultivate.

[0055] 3. After the cells adhere to the wall the next day, observe the well with only one living cell under a microscope and mark it with a marker pen.

[0056] 4. After culturing for 3 days, change the medium in the wells marked with a marker pen.

[0057] 5. After 10 days of culture, the monoclonal grows. Further screen the single clone with faster growth rate and better cell uniformity. Eleven monoclonal cell lines labeled C1 / C2 / C4 / C5 / C6 / C7 / C10 / C11 / C12 / C13 / C14 were obtained. The representative clones are C2 and C10 clones. take pictures as figure 2 .

[0058] 6. When the 96 wells grow to 80% density, digest a...

Embodiment 3

[0060] Example 3: Comparing the calcium phosphate-DNA transfection efficiency and toxicity efficiency of different monoclonals, infecting tumor cells at the same time to verify the infection efficiency and observe and compare the cytotoxicity.

[0061] 1. Cultivate different monoclonal cell lines C1-C14 to the logarithmic growth phase, and plant 4x10 cells in each well of a 12-well plate. 5 cells.

[0062] 2. Mix the GFP-labeled control plasmid with the lentiviral original plasmid, and transfect the plasmid with the calcium phosphate-DNA precipitation method; wherein, the control plasmid is the pCDH-CMV-MCS-EF1-copGFP vector, and the packaged virus is named pCDH- GFP; Viral original plasmids are: Pvsv-G (Clontech: 631530), pCMV delta R8.2 (Addgene: 12263).

[0063] The plasmid vectors used for transfection are: pFUGW-H1 (addgene: 25870) plasmid empty vector and pCDH-CMV-MCS-EF1-copGFP (SBI: CD511B-1) plasmid empty vector.

[0064] 3. After 24 hours of transfection, use a flu...

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Abstract

The invention relates to the field of biotechnology, in particular to a monoclonal cell strain of a human embryo kidney cell 293T-C10 for packaging viruses as well as a screening method of the monoclonal cell strain. 293T cell mycoplasmas are removed, monoclonal screening is carried out during logarithmic growth period to obtain uniform monoclonal cells, different transfection and virus removal efficiencies are compared, and screening is carried out to obtain the monoclonal cell strain. Such cell strain has a collection number of CCTCC C201281, is very high in transfection efficiency and virus packaging stability, is remarkably improved in virus removal efficiency of 293T cells than that before screening, and can infect a majority of tumors and other cells to be infected without ultracentrifugation, so as to meet most of experimental demands.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 293T cell monoclonal cell strain for packaging viruses and a screening method, and the cell strain has high transfection efficiency and stability of virus packaging. Background technique [0002] 293 cells are human renal epithelial cell lines transfected with adenovirus E1A gene. 293T cells are derived from 293 cells and can express SV40 large T antigen. They are mainly used for packaging viruses or transient transfection to overexpress various target proteins. [0003] The types of lentivirus include human immunodeficiency virus HIV, simian immunodeficiency virus SIV, feline immunodeficiency virus FIV, equine infectious anemia EIA, etc., and the culture time is longer. [0004] According to the traditional virus packaging method, the supernatant of 293T cells containing virus particles needs to be concentrated by ultracentrifugation (the method is: after ultracentrifugation, disc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N7/00C12R1/91
Inventor 王海峰姚远颋谈竹君劳昕元
Owner 上海溥生生物科技有限公司
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