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A kind of talen expression vector and its rapid preparation method, target gene and cell double labeling system and application

A technology for expressing vectors and target genes, which is applied in the field of rapid preparation of TALEN expression vectors, target gene and cell double labeling system, to achieve high efficiency and high editing efficiency

Active Publication Date: 2021-07-27
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: In view of the existing gene editing technology and the problems existing in the detection and separation of edited genes and cells, the present invention provides a TALEN expression vector and its rapid preparation method and target gene and cell double labeling system

Method used

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  • A kind of talen expression vector and its rapid preparation method, target gene and cell double labeling system and application
  • A kind of talen expression vector and its rapid preparation method, target gene and cell double labeling system and application
  • A kind of talen expression vector and its rapid preparation method, target gene and cell double labeling system and application

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Embodiment

[0061] TALEN and CRISPR editing of NF-κB family genes in three kinds of cells

[0062] 1. Materials and Methods

[0063] 1.1. Preparation of linear TALEN monomer

[0064] The linear TALEN monomer is used as a template from the plasmid monomer in the "Golden Gate TALEN and TAL Effector Kit 2.0 (purchased from Addgene)" (Addgene kit 2.0 for short; Addgene Kit 2.0) kit, using the ones listed in Table 1 and Table 2. The primers and oligonucleotides were prepared by PCR amplification, and the method was the same as the applicant’s previous patent or literature (invention patent application number: 201810723261.4; Generate TALE / TALEN as Easily and Rapidly as Generating CRISPR Molecular Therapy: Methods & Clinical Development 2019, 13 : 310-320). The prepared 62 linear TALEN monomers were arranged on a 96-well plate as a new PCR template, and a pair of universal primers (PCR-TAL-F / R; Table 1) was used to replicate the single TALEN monomer by 96-well PCR amplification. Body (reprod...

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Abstract

The invention discloses a TALEN expression vector, a rapid preparation method thereof, a target gene and cell double labeling system and application. The present invention can prepare a TALEN expression vector that binds to any 18bp target in one day faster than CRISPR. The invention consists of a set of linear monomers, TALEN backbone plasmids, and adopts a new process to assemble TALEN. The present invention can easily and efficiently obtain ready-to-use TALENs. By preparing five pairs of TALENs and editing NF-κB family genes in three different cell lines, it is proved that the TALEN expression vectors prepared by the present invention are highly efficient, reproducible and reliable. performance and applicability. The TALEN of the present invention has higher editing efficiency than CRISPR. In addition, the invention also discloses a double labeling system for synchronously labeling target proteins and cells through homology-directed repair, which has wide application value in the fields of gene editing and biotechnology.

Description

technical field [0001] The invention relates to the field of gene editing biotechnology, in particular to a rapid preparation technology of a TALEN expression vector, a target gene and cell double labeling system and its application. Background technique [0002] Transcription activator-like effectors (TALEs) are natural transcription activators discovered from bacteria that possess a programmable DNA-binding domain (DBD) consisting of tandem repeats of 34 amino acid sequences (monomers) sequence and repeat variable diresidue (RVD) composition. Through fusion with nuclease FokI, TALE can form a gene editing tool TALEN (ie TALE-FokI). TALEN is a well-known second-generation gene editing tool that is widely used to edit genes. Due to their high targeting properties, TALENs have been used to generate universal chimeric antigen receptor T cells (UCAR-T) for clinical cancer immunotherapy. However, with the birth of CRISPR in 2013, TALEN was completely replaced by CRISPR due to...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/65
CPCC12N15/65C12N15/85C12N15/907C12N2800/107
Inventor 王进科张书衍
Owner SOUTHEAST UNIV