Method for pcr amplified gene nucleotransfer to 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: 2018-02-09
SHANGHAI PUBLIC HEALTH CLINICAL CENT
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for pcr amplified gene nucleotransfer to suspension animal cells
  • Method for pcr amplified gene nucleotransfer to suspension animal cells
  • Method for pcr amplified gene nucleotransfer to suspension animal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Nuclear transfer of PCR amplified gene (PaGene) into Kasumi-1 cells

[0051] The applicant found that the expression plasmid nucleated into Kasumi-1 cells caused a large number of deaths of Kasumi-1 cells; after a large number of experiments, the applicant unexpectedly found that the following methods can significantly reduce the death rate of nucleated Kasumi-1 cells. And to maintain a more ideal cell transfection efficiency and high cell proliferation, so as to provide a feasible nuclear transfer method for subsequent related functional experiments involving Kasumi-1 cell nuclear transfer; the method uses the expression plasmid as a template and uses PCR for amplification purposes Gene (PaGene): The amplified PaGene contains a gene promoter, protein coding region, and transcription terminator; to ensure that PaGene can be successfully transcribed and translated in the cell, then use PaGene to nucleate Kasumi-1 cells.

[0052] This embodiment relates to a method...

Embodiment 2

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

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

[0091] The operation method for nuclear transfer of THP-1 cells is the same as in Example 1, except that the nuclear transfer program of THP-1 cells is V-001.

[0092] PaGene also induces lower cell death and appropriate transfection efficiency in NB4 and THP-1 cells. Since PaGene induces lower cell death and a similar level of transfection efficiency in Kasumi-1 cells, we further explore whether it should 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 are similar to those in Kasumi-1 cells. 72h after nuclear transfection, PaEGFP induced lower cell death in NB4 cells, and even ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
Inventor 吴康范小勇黄家颖赵旭杰
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap