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Method for constructing gene over-expression or interference stable transfer cell strain based on piggyBAC system

A gene overexpression and cell line technology, applied in the field of gene overexpression or interference stable transfection cell line construction based on piggyBAC system, can solve the problems of affecting cell state, affecting endogenous gene expression, genome instability, etc., achieving efficient excision, achieve reversal effect

Inactive Publication Date: 2019-04-16
CYAGEN BIOSCI INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (1) The lentivirus may integrate multiple copies in the genome, which may affect the expression of endogenous genes, and may also cause genome instability;
[0008] (2) Virus infection may affect the state of cells and cause cell aging;
[0009] (3) The carrying capacity of the lentiviral vector is limited, and the packaging titer of the larger gene lentivirus is low, which makes it difficult to construct a cell line;
[0010] (4) The steps are cumbersome: it is necessary to construct a lentiviral vector and carry out virus packaging, and the construction period of a stable transfection is very long;
[0011] (5) Once the gene is integrated, it cannot be removed

Method used

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  • Method for constructing gene over-expression or interference stable transfer cell strain based on piggyBAC system
  • Method for constructing gene over-expression or interference stable transfer cell strain based on piggyBAC system
  • Method for constructing gene over-expression or interference stable transfer cell strain based on piggyBAC system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Construct the overexpression stable transfection strain of gene mCtnnd1[NM_007615.4] in HCT116 cells, and construct the overexpression stable transfection strain by piggyBAC method.

[0051] PiggyBAC method stable transfection strain construction steps:

[0052] pass HiFi DNA Assembly (NEB#E5520) was connected, and the gene overexpression element mCtnnd1[NM_007615.4], fluorescent marker EGFP, and screening resistance gene puromycin were cloned into the piggyBAC expression vector in sequence. The vector information is as follows: figure 2 shown.

[0053] The stable transfection vector and pBase expression vector were co-transfected 1×10 by Lipo3000 5HCT116 cells were screened by adding Puro to the medium 1 day after transfection. After 3 days of screening and culture, active cells were selected for Q-PCR to observe the fluorescence rate of the cells to identify the effect of overexpression or interference of the target gene.

Embodiment 2

[0062] In RAW264.7 cells, the gene mCtnnd1[NM_007615.4] overexpressed stable transfection strain was constructed, and the overexpression stable transfection strain was constructed by piggyBAC method.

[0063] PiggyBAC method stable transfection construction steps:

[0064] pass HiFi DNA Assembly (NEB#E5520) connection method, the gene overexpression element mCtnnd1[NM_007615.4], fluorescent marker EGFP, screening resistance gene Puro were cloned into the piggyBAC expression vector in sequence, the vector information is as follows figure 2 shown.

[0065] The stable transfection vector and the pBase expression vector were co-transfected into RAW264.7 cells 1×10 by Lipo3000 5 Cells were screened by adding Puro to the medium 1 day after transfection. After 3 days of drug screening, the fluorescence rate of the cells was observed.

Embodiment 3

[0075] The hYWHAH[NM_003405.3] overexpression stable transfection strain was constructed in Human MSC cells.

[0076] Try to construct stable transgenic strains by piggyBAC method:

[0077] pass HiFi DNA Assembly (NEB#E5520) was connected, and the gene overexpression element mCtnnd1[NM_007615.4], fluorescent marker EGFP, and screening resistance gene puromycin were cloned into the piggyBAC expression vector in sequence, and the vector was respectively used with Lipo 3000 and Human MSC cells were transfected by electroporation. After 1 day of Puro drug screening for 3 days, the cell state was normal and there was no aging. Electrotransfection is better than Lipo, with more cell survival and fluorescence rate >80%, such as Image 6 shown.

[0078] However, after the lentivirus method infects Human MSC cells at MOI 30, cell aging occurs, and passage and follow-up experiments cannot be performed, and the infection efficiency is very low at low MOI.

[0079] Prove that when t...

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Abstract

The invention discloses a method for constructing a gene over-expression or interference stable transfer cell strain based on a piggyBAC system, which comprises the following steps of: sequentially inserting a gene over-expression or interference element segment, a fluorescent marker segment and a resistance screening segment between ITR segments of a piggyBAC carrier, constructing the carrier, then co-transfecting the constructed vector and the pBase expression plasmid into cells for carrying out resistance screening, selecting resistant survival cells, carrying out Q-PCR detection, transferring pBase-MU plasmid into a positive clone, cutting off the sequence between the ITR segments under the action of mutant pBase, leaving no sequence at the in situ point, after confirmation of successful excision, cryopreserving the positive clone. According to the method, gene overexpression or interference elements are inserted between the ITR segments to construct the piggyBAC stable transfer carrier. No restriction on the size of genes exists, and a larger gene or a plurality of genes can be overexpressed at the same time.

Description

technical field [0001] The invention belongs to the technical field of biological genes, and in particular relates to a method for constructing gene overexpression or interference stably transfected cell lines based on the piggyBAC system. Background technique [0002] The most common method to study the function of a gene is to overexpress or silence the gene in the host cell, and to screen stable cell lines. Stably transformed cell lines play an important role in gene function research, signaling pathway research, protein or antibody expression, and drug development. At present, stable cell lines are mainly constructed by virus method, among which lentivirus is the most commonly used. [0003] Lentiviruses can infect almost all types of cells, and can be integrated into the genome of infected cells after infection for long-term stable expression. Using the characteristics of lentivirus, clone the foreign DNA into a lentiviral vector with certain resistance and infect the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N5/10
CPCC12N5/0602C12N15/65C12N15/85C12N2510/00
Inventor 郑敦武段伟婷程冬洋
Owner CYAGEN BIOSCI INC
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