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A kind of 293t cell strain and its application of stably and efficiently packaging virus

A GC-293T, cell line technology, applied in the field of 293T cell line, can solve the problems that the transfection efficiency cannot meet the production requirements and the titer of the packaging virus is not high.

Active Publication Date: 2018-10-09
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the transfection efficiency of the currently used 293T cells still cannot meet the production requirements, and the virus titer of the packaged virus is not high

Method used

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  • A kind of 293t cell strain and its application of stably and efficiently packaging virus
  • A kind of 293t cell strain and its application of stably and efficiently packaging virus
  • A kind of 293t cell strain and its application of stably and efficiently packaging virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Comparing GC-293T cell line and ATCC 293T cell line (purchased from ATCC, product number is ATCC NO.CRL-3216 TM ) calcium phosphate-DNA transfection efficiency and virus packaging effect, including virus titer and cytotoxicity.

[0051] 2. Inoculate the GC-293T cell line and the ATCC 293T cell line of the present invention on a 6 cm cell culture dish, and cultivate for about 24 hours until the logarithmic growth phase (cell density is about 80%).

[0052] 3. Change the medium before transfection, and take photos and observe with visible light to ensure that the growth status and cell density of the two cell lines are very close.

[0053] 4. Mix the GFP-labeled empty plasmid with the lentiviral element plasmid, and use calcium phosphate-DNA precipitation to transfect GC-293T cell lines and ATCC 293T cell lines with the three-plasmid virus packaging system: Among them, the empty plasmid GV248 (Jikai Gene product article number: PCONGC248028254) the cis-element sequenc...

Embodiment 2

[0063] The plasmids constructed by inserting genes were selected to carry out virus packaging tests and comparisons on GC-293T cell lines and ATCC 293T cell lines.

[0064] 1. Select the plasmid to insert the gene into:

[0065] The GV208-HMOX1 plasmid is a plasmid in which the HMOX1 gene is inserted into the GV208 (Genetic product number: PCON063) vector (the cis element sequence is: Ubiquitin-MCS-EGFP), and the virus packaged on the GC-293T cell line is named GC -293T-HMOX1, the virus packaged on ATCC 293T is named ATCC 293T-HMOX1; the vector map of GV208 is as follows figure 1 shown in .

[0066] 2. GC-293T cell line and ATCC 293T cell line were transfected with calcium phosphate-DNA and transfected with GV208-HMOX1 plasmid. Use 6cm cell culture dishes for transfection and culture.

[0067] 3. Take pictures with fluorescence microscope before and after transfection to compare the transfection efficiency. The results are as follows: Figure 4 . It can be seen that the t...

Embodiment 3

[0072] The transfection efficiency and virus packaging stability of GC-293T cell line were observed by multiple passages.

[0073] The revived GC-293T cell line (marked as P0) was passaged for 12 times, and the cells of different passages were marked as P1-P12.

[0074] Select GC-293T cell lines of P0, P4, and P12 passages for plasmid transfection and virus packaging.

[0075] The selected plasmid: GV248 plasmid; the virus packaged by the plasmid GV248 on the GC-293T cell lines of P0, P4, and P12 passages is named GV248-P0, GV248-P4, and GV248-P12, respectively.

[0076] P0, P4, P12 cell lines were photographed before and after transfection, and the results were as follows Figure 6 . It can be seen that the transfection efficiency of different passages of GC-293T cell lines has no significant difference, that is, with the increase of passage times, the transfection efficiency of cells does not decrease, and remains at 80%.

[0077] Virus GV248-P0, GV248-P4, GV248-P12 infec...

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Abstract

The invention provides a 293T cell line for stably and efficiently packaging a virus and an application thereof, and particularly to a 293T cell line for stably and efficiently packaging the virus, and the preservation number of the cell line is: CCTCC C2015110. The transfection efficiency of the cell line is higher than that of a commercial 293T cell; the virus can be stably and efficiently packaged; moreover, the cell toxicity is lower than that of the commercial 293T cell line; after the cell passes from generation to generation for 12 times, by means of titer detection, the virus titer is always maintained at 1.Oe+9tu / ml or more.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 293T cell strain for stably and efficiently packaging viruses and its application. Background technique [0002] 293 cells are human renal epithelial cell lines transfected with adenovirus E1A gene. 293T cells are derived from 293 cells and express SV40 large T antigen at the same time. The plasmid containing the SV40 replication origin and promoter region can replicate. The transfection efficiency of 293T cells can be as high as 50% with the calcium phosphate transfection method; the protein expression level is high. It is mainly used for virus packaging or transient transfection to overexpress various foreign target proteins. [0003] Currently, the American Type Culture Collection (ATCC) has developed this cell line into commercialization, and it is widely used for in vitro detection and production of recombinant proteins and viruses. However, the transfection efficiency of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073C12N5/0783C12N7/00C12N5/10C12R1/93
Inventor 吴涛姜军高博谢胜华朱向莹曹跃琼
Owner SHANGHAI GENECHEM
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