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Single-input multi-output gene loop, expression vector, host cell and application of host cell

A single-input, multiple-output, host cell technology, applied to cells modified by introducing foreign genetic material, vectors, using vectors to introduce foreign genetic material, etc., to achieve the effects of promoting proliferation and differentiation, avoiding accidental injury, and promoting proliferation and/or differentiation

Inactive Publication Date: 2019-08-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The results of gene circuits in eukaryotic cells usually use AND / OR / NO logic gates to achieve multiple-input and multiple-output results. There is no scheme to produce multiple output results based on changes in the intensity of a single input signal.

Method used

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  • Single-input multi-output gene loop, expression vector, host cell and application of host cell
  • Single-input multi-output gene loop, expression vector, host cell and application of host cell
  • Single-input multi-output gene loop, expression vector, host cell and application of host cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construct a screening system based on the ability to suppress downstream expression of fluorescent proteins to screen repressible promoters with good orthogonality and their binding DNA sequences.

[0049] The screening system is as follows: the upstream is the gene expressing the transcription factor and the gene expressing the green fluorescent protein EFGP, and the downstream is the synthetic promoter containing the transcription factor combined with DNA and the gene expressing the red fluorescent protein mCherry.

[0050] The transcription factors PIP and ZF43-8 and their binding DNA were detected separately: the downstream synthetic promoter of PIP contained 4 copies of PIP binding DNA sequence (SynPro(S)-PIP; 4, figure 1 A); The downstream synthetic promoter of ZF43-8 contains 8 copies of the ZF43-8 binding DNA sequence (SynPro(S)-ZF43-8; 8*, figure 1 B). The results showed that: for the repressive transcription factor (SynPro(S)) with repressive ability, with it...

Embodiment 2

[0060] Construct a gene circuit with one input and multiple outputs (three outputs), the gene circuit includes 1 module, each module includes 3 submodules, that is, 3 independent transcription units, each transcription unit is regulated by a promoter, and can be Simultaneous expression of 1-3 different peptide chains. Such as image 3 As shown, the gene circuit is mainly composed of three submodules:

[0061] The 1.1st submodule unit includes an activating promoter, a repressible transcription factor Y coding sequence (represented by SynTF Y), a repressive transcription factor X coding sequence (represented by SynTF X1) and functional protein C (the first functional protein); There is good orthogonality and significant difference in repression threshold between Y and X, and the repression threshold of repressed transcription factor Y is smaller than that of X;

[0062] The 1.2 submodule includes the coding sequence of the repressible synthetic promoter SynPro Y, the repressi...

Embodiment 3

[0073] After the amino acid sequence of the target cell surface antigen (immune protein or transmembrane protein with obvious extracellular fragments) and the amino acid sequence of the corresponding single-chain antibody are known, different states (such as cells with tumor lesions, infection or mutations / Normal cells) on the target cell surface antigen density, and screen to obtain intracellular signal substances corresponding to changes in antigen density, such as mediated by transmembrane proteins; and select differential response signal input strengths (thresholds are different and orthogonal performance is good ) transcription factor and its binding DNA binding sequence and copy number to construct a single-input and three-output gene circuit, the structure of which is the same as in Example 2.

[0074] The repressive transcription factor Y is PIP, and the binding DNA on SynPro Y is 4 copies of 5-GAAATAGCGCTGTACAGCGTATGGGAATCTCTTGTACGGTGTACGAGTATCTTCCCGTACACCGTAC-3.

[...

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Abstract

The invention relates to the technical field of biology, and discloses a single-input multi-output gene loop. The gene loop selects two pairs of inhibitory transcription factors having good orthogonality and remarkable inhibition efficiency differences and DNAs combined with the inhibitory transcription factors for responding to the cell surface antigen density, and different functional proteins are driven to be expressed according to the intensity of input signals. Effect cells are improved through the single-input multi-output gene loop, and can be driven to differentially express differentfunctional proteins such as cell factors along with density changes of antigens of target cells, and a combination effect on the target cells is achieved. According to the single-input multi-output gene loop, the efficiency for cell treatment of a single antigen can be improved, and the single-input multi-output gene loop is considered in a method for cell treatment of multiple antigens.

Description

technical field [0001] The invention belongs to the field of biotechnology, especially a gene circuit that can produce different responses according to the intensity of a single signal input, and use the gene circuit to transform host cells so that it can produce different responses to different densities of single antigens on the cell surface. Background technique [0002] Cancer is difficult to cure due to the endogenous, heterogeneous and mutational drug resistance of cancer cells. Chimeric antigen receptor (CAR), also known as artificial antigen receptor (artificial antigen receptor), is a type of artificially modified, composed of extracellular antigen binding domain, transmembrane domain and intracellular signal transduction domain , a T cell receptor that specifically recognizes and targets tumor antigens and activates T cells. T cells expressing CAR are referred to as CAR-T. T cells recognize specific antigens on the surface of tumor cells through CAR, that is, spe...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C07K14/52C12N2830/34
Inventor 蔡亮杨子杰
Owner FUDAN UNIV
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