Method for pcr amplified gene nucleotransfer to suspension animal cells

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 specifically relates to a method for nuclear transfer of suspension animal cells for PCR amplification of genes. Background technique [0002] Nucleic acid transfected cells (small non-coding RNA, messenger RNA, or plasmid) are widely used in functional genomics research, vaccine research, transcription regulation research, stem cell research, inflammatory disease treatment research, etc.; used in cell lines or primary generation Cell transfection methods can be divided into two categories: one is virus-based transfection methods; the other is non-virus-based transfection methods. For transfection methods that are not based on viruses, electrophoration is one of the most important methods (especially for suspension cells). The basic principle of electrophoresis is that the plasma membrane of the cell is briefly permeated by electric shock so that nucleic acid can enter the cell. . As an upgraded ver...

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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 Objective and accurate interpretation of the biological effect of the transfected target gene, a reliable nuclear transfer method for Kasumi-1 cells is urgently needed in the prior art, so as to carry out relevant functional experiments involving Kasumi-1 nuclear transfer

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