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Modular construction of synthetic gene route in mammalian cell by TALE transcription suppressor

A cell, coding gene technology, applied in the field of modular construction of synthetic gene circuits, can solve problems such as low inhibition efficiency

Active Publication Date: 2015-05-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that in the context of mammalian gene regulation, placing the LacI binding site downstream of the cytomegalovirus promoter (CMV) or CAG promoter in the synthetic gene circuit can inhibit gene expression, although the inhibition efficiency is lower than that of mammals. Animal expression system is lower than prokaryotic expression system

Method used

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  • Modular construction of synthetic gene route in mammalian cell by TALE transcription suppressor
  • Modular construction of synthetic gene route in mammalian cell by TALE transcription suppressor
  • Modular construction of synthetic gene route in mammalian cell by TALE transcription suppressor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] The method of transfecting cells with plasmids in Example 1 and Example 2: take a 24-well plate and inoculate 0.5 mL of HEK293 cell suspension (containing 6×10 4 HEK293 cells), cultured for 24 hours, replaced with new DMEM medium, and then carried out plasmid transfection.

[0127] Example 1, Functional verification and specificity analysis of TALER protein

[0128] The schematic diagram of the action mechanism of pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid is shown in figure 2 . Under the action of the pEF1a promoter, TagBFP and Gal4 / vp16 are expressed (the 2A connecting peptide between TagBFP and Gal4 / vp16 is a self-splicing peptide, so TagBFP can represent the expression level of Gal4 / vp16). Gal4 / vp16 activated the 5×UAS sequence, thereby activating the transcriptional initiation of the CMVmini promoter, and mKate2 was expressed. Under the action of the CMV promoter, EYFP and TALER1 proteins are expressed (the 2A connecting peptide between EY...

Embodiment 2

[0195] Embodiment 2, further ductility research

[0196] The pEF1a-TagBFP-2A plasmid is the pEF1a-TagBFP-2A plasmid in Example 1.

[0197] The pCMV-TALER1 plasmid is the pCMV-TALER1 plasmid in Example 1.

[0198] The pCMV-TALER2 plasmid is the pCMV-TALER2 plasmid in Example 1.

[0199] The pCMV-TALER4 plasmid is the pCMV-TALER4 plasmid in Example 1.

[0200] The pCMV-TALER5 plasmid is the pCMV-TALER5 plasmid in Example 1.

[0201] The pCMV-TALER32 plasmid is the pCMV-TALER32 plasmid in Example 1.

[0202] The pT1+T1+72-DsRed plasmid is shown in SEQ ID NO:54. In sequence 54, nucleotides 2441-2533 from the 5' end are 5×UAS sequences, nucleotides 2549-2562 are T1 sequences (target sequence of TALER1 protein), nucleotides 2569-2628 It is a CMVmini promoter, the 2635-2648th nucleotide is the T1 sequence, and the 2668-3345th nucleotide is the coding gene of DsRed (red fluorescent protein).

[0203] The pT1+T2+72-DsRed plasmid is shown in SEQ ID NO:55. In sequence 55, nucleoti...

Embodiment 3

[0245] Example 3, Modular construction of the gene circuit of the TALER protein cascade

[0246] The pCAG-rtTA-2A-Gal4 / vp16 plasmid is shown in SEQ ID NO:73. In sequence 73, nucleotides 4253-4930 from the 5' end are the CAG promoter, nucleotides 6004-6747 are the gene encoding rtTA, and nucleotides 6748-6813 are the gene encoding the 2A linker peptide , the 6820-7503 nucleotides are the coding gene of Gal4 / vp16.

[0247] The pT14+T14+72-mKate2 plasmid is shown in SEQ ID NO:74. In sequence 74, nucleotides 4275-4367 from the 5' end are 5×UAS sequences, nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein), nucleotides 4406-4465 It is a CMVmini promoter, the 4472-4488th nucleotide is the T14 sequence, and the 4538-5243rd nucleotide is the coding gene of mKate2. The pT14+T14+72-mKate2 plasmid has two TALER14 protein binding sites (pT14BS2).

[0248] The pT14+T14x3+72-mKate2 plasmid is shown in SEQ ID NO:75. In sequence 75, nucleotides 69-161 from the 5'...

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Abstract

The invention aims to provide modular construction of a synthetic gene route in a mammalian cell by a TALE (transcription activator-like effector) transcription suppressor. According to a protected method for realizing regulatable expression of two proteins involved in the invention, an expression cassette A includes: a coding sequence of a feedback element, a promoter A, protein A's coding gene and TALER protein A's coding gene that are connected through self-shearing polypeptide, and a target sequence A (including the target sequence of shRNA1); an expression cassette B includes: a coding sequence of a feedback element, a promoter B, protein B's coding gene and TALER protein B's coding gene that are connected through self-shearing polypeptide, and a target sequence B (including the target sequence of shRNA2); and an expression cassette C includes a constitutive promoter and a coding sequence of an activating element. A recombinant vector A with the expression cassette A, a recombinant vector B with the expression cassette B and a recombinant vector C with the expression cassette C are imported into a host cell, and shRNA1 or shRNA2 is added to regulate expression of protein A and protein B.

Description

technical field [0001] The present invention relates to the modular construction of synthetic gene circuits in mammalian cells using TALE transcriptional repressors. Background technique [0002] The synthetic gene circuit is carefully designed, and the gene regulation device is assembled according to the function, and the molecular information in the cell is sensed, integrated, and processed to perform certain functions. Various synthetic gene circuits have been developed to achieve customizable, programmable functions within cells, including dynamic behavior, switching and memory, intercellular communication, fitness, cell polarization, digital and analog computation, and complex biosynthetic pathways. Most of these genetic circuits were constructed using limited genetic elements and costly, inefficient "trial-and-error" methods. Therefore, to simplify design and optimize complex manipulations in living cells, it is necessary to develop a large-scale library of functional...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/63C12N15/64
Inventor 谢震威斯李寅青蒋云廖微曦陈赫
Owner TSINGHUA UNIV
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