Transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency

A eukaryotic cell and non-viral technology, applied in the genetic field, can solve the problems of high gene cloning technology and time requirements, low stable transfection efficiency, etc., and achieve the effect of simplifying technology and time, and improving efficiency

Active Publication Date: 2020-01-31
NANTONG UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the stable transfection efficiency of the dual-vector SB system is low, and the technology and time required for gene cloning are high

Method used

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  • Transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency
  • Transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency
  • Transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Transfection and detection of immortalized human primary bronchial epithelial cells (HBEC)

[0049] (1-1) Collect the immortalized HBEC cells grown in several phases, resuspend in 100ul Lonza SF transfection reagent (Lonza), add to the transfection cup, the cell concentration is 10 7 / ml. Add ITR-CMV-GFP vector and EF1-SB100X (2ug+1ug, ratio 2:1, plasmid concentration greater than 0.5ug / ml), or SB-2in1-GFP (3ug, plasmid concentration greater than 0.5ug / ml) flick transfection Mix the staining cup and let it stand for 5 minutes. In the control group, only 2ug ITR-GFP carrier was added.

[0050] (1-2) The CM-130 program of Lonza 4D-Nucleofector (Lonza) was used for electrotransfection of plasmids.

[0051] (1-3) After transfection, add 500ul of medium to the electroporation cup, and use the disposable pipette provided in the kit to add all the cell suspension (about 600ul) into 15ml of complete medium (RPMI1640+10% fetal bovine serum ) in T75 cell culture fla...

Embodiment 2

[0054] Such as Figure 6 Shown is the high-efficiency stable transfection of HBEC cells induced by the SB single-vector system: the proportion of GFP-positive cells detected by flow cytometry after 2 and 10 days of HBEC cell transfection. The results showed that although the efficiencies of transient transfection in the three groups reached about 90% (Day 2), after 10 days (Day 10), the GFP-positive cells in the ITR-CMV-GFP alone transfection group were less than 1%; However, about 20% of the cells were still positive for GFP after transfection with the SB double plasmid. In contrast, after 10 days of expansion of cells transfected with a single plasmid of SB-2in1-GFP, the positive rate of GPF was more than 50%, which proved that the efficiency of stable transfection was significantly improved. Example 2: Transfection and detection of human lung cancer cell line PC-9

[0055] (1-1) Collect PC9 cells grown in several phases, resuspend in 100ul Lonza SE transfection reagent, a...

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Abstract

The invention provides a transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency. The method is characterized in that the transposon mediated stable transfectionefficiency is improved in an eukaryocyte while an experimental operation is simplified by constructing a vector system which can simultaneously express a target gene and a transposase SB100X. More specifically, a single vector SB-2in1-DEST is constructed, and GFP is used as a target gene for testing, so that the cell proportion of stable transfection GPF is increased by about 2.5 times that of a transposon double delivery system, which proves that the transfection method can remarkably improve the efficiency of non-viral stable transfection. Furthermore, the target gene can be conveniently cloned into the SB-2in1-DEST vector constructed by the method by adopting a Gateway system, and the technique and time for cloning can be simplified.

Description

technical field [0001] The invention belongs to the field of gene technology, and in particular relates to a transposon-mediated high-efficiency non-viral eukaryotic cell stable transfection method. Background technique [0002] There are two types of stable transfection of eukaryotic cells, virus and non-virus. Although the method of using virus as a carrier has high efficiency, the size of the gene carried is limited, and because the virus is potentially carcinogenic, it is a significant safety hazard. The advantage of non-viral vectors is good safety, but the disadvantage is low efficiency. The ideal eukaryotic cell transfection technology should be safe and efficient at the same time, but the current methods are insufficient, which is the current technical bottleneck of eukaryotic cells, especially primary eukaryotic cell gene modification. [0003] Transposon (Transposon) is a kind of DNA segment, which can be inserted into other parts of genomic DNA under the action o...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N15/85
Inventor 周晓荣丁晓凌汪晓莺
Owner NANTONG UNIVERSITY
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