RU2630654C2 - Method for neurogenesis chemogenetic recording and correction based on genetic constructions for astrocytes and neurons transfection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is a method for chemogenetic recording and correction of neurogenesis based on genetic constructs for astrocytes and neurons transfection, in which a chemical compound having activity of neural tissue stimulation and formation and/or neuronal degeneration inhibition is used, Where genetic constructs are developed based on the lentiviral vector for specific transfection of astrocytes and neurons shown in Fig. 2 and Fig. 3, which have the ability to stimulate or inhibit targeted genes expression in astrocytes and neurons populations, and also to express a fluorescent marker therein.

EFFECT: invention allows cell-specific transfection of various populations of neural tissue cells, in particular astrocytes and neurons, to inhibit and/or activate neurogenesis, and to visualize the transfected cells in vitro by integrating fluorescent markers into the genetic construct under construction.

3 dwg

Description

The invention relates to the field of molecular biology for the registration and correction of neurogenesis through specific transfection of astroglia cells with the aim of targeted monitoring of intracellular cascades, in particular for the study of neurodegenerative diseases, and can also be used in the field of medicine for the treatment of neurodegenerative diseases.

The invention is known "Method and composition for stimulating neurogenesis and inhibiting neuronal degeneration" (RF patent No. 2440346), containing a chemical compound similar to that used in the claimed method, having the ability to stimulate neurogenesis and (or) inhibit neuronal degeneration.

The disadvantage of this method is that using the chemical compound described in it, it is not possible to stimulate or inhibit the expression of target genes in populations of brain neurons, as well as to express a fluorescent marker in neurons.

The closest in technical essence of the claimed method, selected as a prototype, is the "Method and composition for stimulating neurogenesis and inhibiting neuronal degeneration" (RF patent No. 2440346 01/20/2012), which uses a chemical compound with activity to stimulate the formation of nervous tissue and ( or) inhibiting neuronal degeneration.

The disadvantage of this method is the inability to stimulate or inhibit the expression of target genes in populations of astrocytes and brain neurons, as well as the expression of a fluorescent marker in neurons.

The objective of the invention is the implementation of the mechanism of targeted monitoring of intracellular cascades in astrocytes in order to stimulate neurogenesis and (or) inhibition of neuronal degeneration, in particular, to study neurodegenerative diseases by specific transfection of astroglia and neurons with genetic constructs that allow visualization of certain types of brain cells using fluorescent markers.

The problem is solved in that in a method of chemogenetic registration and correction of neurogenesis based on genetic constructs for transfection of astrocytes and neurons, in which a chemical compound with activity of stimulation and formation of nervous tissue and (or) inhibition of neuronal degeneration is used, according to the invention, genetic constructs are developed based on a lentiviral vector for specific transfection of astrocytes and neurons with the ability to stimulate or inhibit exp target genes in astrocyte and neuron populations, as well as expressing a fluorescent marker in them.

To effectively inhibit the expression of target genes, it is envisaged to integrate cell-specific promoters into the sequence of the genetic construct that ensure the synthesis of genes of interest only in a certain type of cell.

For controlled stimulation of neurogenesis and (or) inhibition of targeted genes, it is envisaged to integrate genes into the sequence of the genetic construct, the expression of which is activated under the influence of chemical stimuli, which, in turn, leads to an increase in the concentration of certain substances that affect the release of glyotransmitters, which, in turn, in turn, they affect the vital activity of neurons and nerogenesis, in particular, as well as providing the ability to visualize individual brain cells about the brain due to fluorescence.

Moreover, in the closest in technical essence of the claimed method uses a chemical compound that is different in structure from the viral vector and is not able to integrate into the genome of brain cells.

In FIG. 1 is a schematic representation of a genetic construct based on a lentiviral vector specifically transfecting astrocytes and (or) neurons. Genetic construction contains:

LTR - long terminal repeat of lentivirus;

GAL4 is a chimeric transactivator consisting of a portion of the transactivation domain of the murine protein NF-κBp65 fused to the DNA-binding domain of the GAL4 yeast protein;

CMV is the minimal promoter of human cytomegalovirus;

GFAP - a compact promoter of glial fibrillar acidic protein;

SYN is the promoter of human synapsin-1;

Case12 - green fluorescent Ca ²⁺ -sensitive protein;

WPRE is the post-transcriptional regulatory element of the marmot hepatitis virus.

In FIG. 2 is a schematic representation of a genetic construct for specific transfection of astrocytes. The genetic design of the LVV-Astro-Case12 contains:

5 'LTR (1-25) - a long terminal repeat of lentivirus;

RRE - rev-responding element;

GAL4 is a chimeric transactivator consisting of a portion of the transactivation domain of the mouse protein NF-κBp65 fused to the DNA-binding domain of the GAL4 yeast protein;

CMV - promoter of human cytomegalovirus;

GFAP - a shortened promoter of a cell-specific marker of glial fibrillar acid protein (astrocyte-specific promoter);

Case12 - green fluorescent Ca ²⁺ -sensitive protein;

MCS is a multiple cloning site;

WPRE - posttranscriptional regulatory element of the marmot hepatitis virus;

11781 bp is the length of the plasmid sequence equal to 11781 nucleotides.

In FIG. 3 is a schematic representation of a genetic construct for specific transfection of astrocytes. The genetic design of LVV-Neuron-EGFP contains:

RRE - rev-responding element;

GAL4 is a chimeric transactivator consisting of a portion of the transactivation domain of the mouse protein NF-κBp65 fused to the DNA-binding domain of the GAL4 yeast protein;

CMV - promoter of human cytomegalovirus;

SYN - human synapsin-1 promoter (neuron-specific promoter);

EGFP - green fluorescent protein; MCS is a multiple cloning site;

WPRE - posttranscriptional regulatory element of the marmot hepatitis virus;

11100 bp is the length of the plasmid sequence equal to 11100 nucleotides.

The method is as follows.

The claimed method is based on the use of a cell-specific promoter to control the simultaneous expression of the desired transgene and a strong artificial transcription activator to enhance transcription by binding to specific binding sites introduced into the promoter sequence. Thus, two cell-specific promoters are involved: one for transcribing the transgene of interest, and the other for transactivator expression.

The genetic constructs used are a multidirectional lentiviral vector system containing the human synapsin-1 promoter (SYN, cell-specific promoter for neurons) and the promoter of the compact glial fibrillar acid protein (GFAP, cell-specific promoter for astrocytes) enhanced in terms of transcriptional activity, and also in the opposite orientation, the promoter derived from human cytomegalovirus (CMV), located in the nucleotide sequence above the cell-specific promoters. Thus, synthetic multidirectional promoters are created, flanked by two genes of the expression cassette. An artificial transcription activator transcribes the 5'-end of the cassette. The sequence of the cassette located along the transcription controls the synthesis of the gene of interest.

The genetic construct for specific transfection of astrocytes (Fig. 2) is constructed using a lentiviral vector based on self-inactivated HIV and includes at least

rev-response element (RRE),

a chimeric transactivator consisting of a portion of the transactivation domain of the murine protein NF-κBp65 fused to the DNA-binding domain of the yeast protein GAL4 (GAL4),

human cytomegalovirus (CMV) promoter in the opposite orientation,

a shortened promoter of a cell-specific marker of glial fibrillar acid protein (GFAP) for transfection of astrocytes,

genetically encoded calcium indicator - Case12 as a marker gene,

multiple cloning site (MCS),

post-transcriptional regulatory element of marmot hepatitis virus (WPRE) to increase vector titer and improve transgene expression.

The genetic construct for specific transfection of neurons (Fig. 3) is constructed using a lentiviral vector based on self-inactivated HIV and includes at least

rev-response element (RRE),

a chimeric transactivator consisting of a portion of the transactivation domain of the murine protein NF-κBp65 fused to the DNA-binding domain of the yeast protein GAL4 (GAL4),

human cytomegalovirus (CMV) promoter in the opposite orientation,

human synapsin-1 promoter (SYN) for specific transfection of neurons,

fluorescent protein - EGFP as a marker gene,

multiple cloning site (MCS),

post-transcriptional regulatory element of marmot hepatitis virus (WPRE) to increase vector titer and improve transgene expression.

The production of genetic constructs is carried out in a HEK293FT cell culture by transient cotransfection of the engineered genetic construct, the pNHP packaging vector, and the pHEF-VSVG plasmid encoding the envelope.

Transfection of cultures of primary astrocytes and neurons obtained from three-day-old rat pups and grown for astrocytes - on Dulbecco's modified Eagle medium (DMEM) (with 4.5 g / l glucose, glutamine, and pyruvate) with 10% fetal bovine serum added, for neurons - on Dulbecco's modified Eagle’s medium (DMEM) (with 4.5 g / l glucose, glutamine, and pyruvate) supplemented with 10% fetal bovine serum and 5% horse serum, as follows: the day before transfection, the cells were plated in 24-well density culture tablets Strongly cells with 5 × ¹⁰ April per well and incubated for 8-14 hours, after which neurons and astrocytes transfected with genetic constructs designed (each corresponding design culture) in the presence of polybrene (8 ug / ml), washed with PBS and cultured for 40 -52 hours in a DMEM environment.

Using the claimed invention allows cell-specific transfection of various populations of cells of the nervous tissue, in particular, astrocytes and neurons in order to inhibit and (or) activate neurogenesis, as well as visualize in vitro transfected cells by incorporating fluorescence markers into the developed design.

Claims (1)

A method of chemogenetic registration and correction of neurogenesis based on genetic constructs for transfection of astrocytes and neurons, which uses a chemical compound with activity of stimulation and formation of neural tissue and / or inhibition of neuronal degeneration, characterized in that they develop genetic constructs based on a lentiviral vector for specific transfection astrocytes and neurons shown in FIG. 2 and FIG. 3, with the ability to stimulate or inhibit the expression of targeted genes in populations of astrocytes and neurons, as well as express a fluorescent marker in them.

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