Method for PCR (polymerase chain reaction) amplification of gene nucleofection of suspension animal cells

A technology for animal cells and gene amplification, applied in the biological field, can solve problems such as low transfection efficiency and cell death, and achieve the effect of reducing mortality

Active Publication Date: 2014-04-23
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Application Information

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Problems solved by technology

However, the researchers found in the routine nuclear transfer practice of Kasumi-1 cells that nuclear transfer with plasmids always induced more severe cell death and lower transfection efficiency, and the high death rate caused by plasmid nuclear transfer may affect...

Method used

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  • Method for PCR (polymerase chain reaction) amplification of gene nucleofection of suspension animal cells
  • Method for PCR (polymerase chain reaction) amplification of gene nucleofection of suspension animal cells
  • Method for PCR (polymerase chain reaction) amplification of gene nucleofection of suspension animal cells

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1, PCR amplified gene (PaGene) nuclear transfer to Kasumi-1 cells

[0051] The applicant found that after the expression plasmid was nuclear-transferred to Kasumi-1 cells, it caused a large number of Kasumi-1 cells to die; after a large number of experiments, the applicant unexpectedly found that the death rate of nuclear-transferred Kasumi-1 cells could be significantly reduced after the following methods were used, And maintain ideal cell transfection efficiency and higher cell proliferation, so that for the relevant functional experiments related to Kasumi-1 cell nuclear transfer in the follow-up, a feasible nuclear transfer method is provided; the method uses the expression plasmid as a template, and the purpose is amplified by PCR. Gene (PaGene); the amplified PaGene contains gene promoter, protein coding region, and transcription terminator; ensure that PaGene can be successfully transcribed and translated in cells, and then use PaGene to nucleotransfer K...

Embodiment 2

[0089] Example 2. Nuclear transfer of PCR amplified gene (PaGene) to NB4 cells and THP-1 cells

[0090] The operation method of nuclear transfer to NB4 cells is the same as that in Example 1, except that the nuclear transfer program of NB4 cells is X-001;

[0091] The operation method of nucleotransfer to THP-1 cells is the same as that in Example 1, except that the procedure of nucleotransfer to THP-1 cells is V-001.

[0092]PaGene also induced lower cell death and appropriate transfection efficiency in NB4 and THP-1 cells, since PaGene induced lower cell death and similar level of transfection efficiency in Kasumi-1 cells, we further explored whether This phenomenon is also true in other suspension animal cell lines. We chose two other suspension cell lines (NB4 and THP-1) for further nuclear transfer experiments. The results in these two cell lines were similar to those in Kasumi-1 cells. 72h after nucleotransfer, PaEGFP induced lower cell death and even higher transfe...

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Abstract

The invention relates to a method for PCR (polymerase chain reaction) amplification of gene nucleofection of suspension animal cells, belonging to the field of biotechnology. The method comprises the following steps of designing PCR primers by taking the expression plasmid of a recombination target gene as a template, wherein a forward PCR primer is positioned at the upstream part of a plasmid promoter (the target gene is corresponding to the promoter), and an inverse PCR primer is positioned at the downstream part of a plasmid transcription terminator (the target gene is corresponding to the terminator); performing PCR amplification through the designed PCR primer by taking the expression plasmid of the recombination target gene as a PCR template; recycling a PCR product; selecting the PCR product, and performing cell transfection according to the standard operation procedure of a nucleofection kit of the Lonza company. Compared with the nucleofection plasmid, PaGene nucleofection can remarkably reduce the death rate of the suspension animal cells; the nucleofection efficiency is not remarkably reduced or is reduced a little while ensuring relatively low death rate of the suspension animal cells after nucleofection by the PaGene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for nuclear transfer of PCR amplified genes to suspended animal cells. Background technique [0002] Nucleic acid transfection cells (non-coding small RNA, messenger RNA, or plasmid) are widely used in functional genomics research, vaccine research, transcriptional regulation research, stem cell research, inflammatory disease treatment research, etc.; for cell lines or primary Cell transfection methods can be divided into two categories: one is virus-based transfection methods; the other is non-virus-based transfection methods. For non-virus-based transfection methods, electrophoration is one of the most important methods (especially for suspension cells). The basic principle of electroporation is to briefly permeabilize the plasma membrane of cells by electric shock, so that nucleic acids can enter cells . As an upgraded version of electroporation, Nucleofecti...

Claims

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Application Information

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IPC IPC(8): C12N15/87
Inventor 吴康范小勇黄家颖赵旭杰
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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