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
CN109628488AInactive Publication Date: 2019-04-16CYAGEN BIOSCI INC +1
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
CYAGEN BIOSCI INC
Publication Date
2019-04-16
Estimated Expiration
Not applicable · inactive patent

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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.
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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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