RU2633691C2 - Method for genetic control of exocytosis based on genetic constructions for astroglia cells transfection - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a genetic construct for the method for the genetic control of exocytosis. The presented construction has the sequence of SEQ ID NO:4 and is obtained using a lentiviral vector based on HIV-1 modified by sequences encoding a post-transcriptional control element of marmot hepatitis B virus (WPRE), a genetically encoded calcium indicator (GCaMP3) and a cell specific promoter of glial fibrillar acidic protein (GFAP).

EFFECT: invention allows to control exocytosis through specific transfection of astroglial cells and to visualize in vitro transfected cells by embedding the genetically encoded calcium indicator in the developed genetic construct and can be used in medicine, in particular for the study of neurodegenerative diseases.

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Description

Technical field

The invention relates to the field of molecular biology for the genetic control of exocytosis through specific transfection of astroglia cells with the aim of controlling vesicular transport of astroglia, in particular for the study of neurodegenerative diseases, can be used in the field of medicine for the correction of neurodegenerative diseases.

State of the art

The invention is known "Peptides that inhibit neural exocytosis" (RF patent No. 2461568), which uses a chemical compound that has the ability to regulate neural exocytosis.

The disadvantage of this method is that, using the chemical compound (peptide) described in it, it is not possible to control the vesicular transport of astroglia, stimulate or inhibit the expression of certain (target) genes in populations of brain astroglia, and also to express a genetically encoded calcium indicator in astroglia cells .

The invention is known “Improvement of genetic constructs to increase the effectiveness of antiviral therapy” (RF patent No. 2533817), which uses a chemical compound similar in nature to that used in the claimed method, having antiviral activity against human immunodeficiency virus.

The disadvantage of this method is that it is not possible to control exocytosis using the chemical compound described in it (genetic construction), as well as to stimulate or inhibit the expression of certain (target) genes in brain astroglia cell populations, and to express a genetically encoded calcium indicator in astroglia cells.

The closest in technical essence of the claimed method, selected as a prototype, is the invention "Peptides that inhibit neural exocytosis" (RF patent No. 2461568, 2007, C07K 14/705, C07K 14/47, C07K 7/06, C07K 7 / 08, A61K 8/64, A61Q 19/08), which uses a chemical compound that has the ability to regulate neural exocytosis, as well as treat conditions requiring the regulation of neural exocytosis, such as, for example, muscle spasticity, facial asymmetry and / or wrinkles on face.

The disadvantage of this method is the inability to control the vesicular transport of astroglia, stimulation or inhibition of the expression of target genes in populations of brain astroglia cells, as well as the expression of a genetically encoded calcium indicator in them.

Disclosure of invention

The objective of the invention is the implementation of a mechanism of targeted genetic control of exocytosis by specific transfection of astroglia cells with the aim of controlling vesicular transport of astroglia, in particular for the study of neurodegenerative diseases, can be used in the field of medicine for the correction of neurodegenerative diseases.

The problem is solved in that in the method of genetic control of exocytosis based on genetic constructs for transfection of astroglia cells, in which a chemical compound capable of controlling exocytosis based on the modification of cerebral astroglia cell vesicles is used to create a new type of therapy for neurodegenerative diseases, according to the invention, develop a method for the genetic control of exocytosis based on a genetic construct for transfection of astroglia cells with the ability to carry out targeted monitoring of intracellular cascades associated with astroglial vesicular transport, as well as to express a genetically encoded calcium indicator in them.

For effective targeted control of intracellular cascades associated with astroglial vesicular transport, it is envisaged to integrate a cell-specific promoter into the sequence of the genetic construct, which ensures the synthesis of genes of interest only in a certain type of cells.

For targeted control of the intracellular cascades associated with astroglial vesicular transport, 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 gliotransmitters, which in turn, they affect the vital activity of neurons, as well as providing the ability to visualize individual brain cells 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.

Brief Description of the Drawings

In FIG. 1 is a schematic representation of a genetic construct based on a lentiviral vector specifically transfecting astroglia cells for genetic control of exocytosis. A genetic construct for transfection of astroglia cells for the genetic control of exocytosis contains:

LTR - long terminal repeat of lentivirus;

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 is the minimal promoter of human cytomegalovirus;

GFAP - a compact promoter of glial fibrillar acidic protein;

GCaMP3 - a sequence encoding a genetically encoded calcium indicator GCaMP3;

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

In FIG. 2 presents a map of the plasmid LVV-GFAP-GCaMP3, characterized by:

5 'LTR (1-75) - long terminal repeat of lentivirus;

SV40 poly (A) signal (75-196) - SV40 polyadenation signal;

GAL4 (1045-1485) is a chimeric transactivator consisting of a portion of the transactivator domain of the mouse protein NF-κBp65 fused to the DNA-binding domain of the GAL4 yeast protein;

CMV (1586-1624) - promoter of human cytomegalovirus;

GFAP (1743-2425) - a shortened promoter of the cell-specific marker of glial fibrillar acidic protein (astroglia-specific promoter);

GCaMP3 (2448-3920) - a sequence encoding a genetically encoded calcium indicator GCaMP3;

ECFP (2748-3017) - enhanced blue fluorescent protein;

EGFP (3039-3470) - enhanced green fluorescent protein;

Calmodulin (3477-3920) - calcium binding protein;

WPRE (3923-4540) - the post-transcriptional regulatory element of the marmot hepatitis virus;

bGH poly (A) signal (4741-4965) - polyadenylation signal;

ori (6309-6897) is a high-copy replication start point for plasmids Co1E1 / pMB1 / pBR322 / pUC;

AmpR (7068-7928) - a sequence that provides resistance to ampecillin, carbencillin and other related antibiotics;

AmpR promoter (7929-8033) - a promoter that provides resistance to ampicillin;

RRE (8534-8767) is a rev-responsive HIV-1 element that provides Rev-dependent export of mRNA from the nucleus to the cytoplasm;

3 'LTR (8768-9186) - a long terminal repeat of lentivirus;

9186 bp is the length of the plasmid sequence equal to 9186 nucleotides.

The implementation of the invention

The method is as follows.

The claimed method is based on the use of genetic constructs for transfection of astroglia cells carrying 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 used genetic constructs are a multidirectional lentiviral vector system (Fig. 1), which contains the promoter of the compact glial fibrillar acid protein (GFAP, cell-specific promoter for astroglia cells) enhanced in terms of transcriptional activity, as well as the promoter obtained from human cytomegalovirus in the opposite orientation (CMV) located in the nucleotide sequence above the cell-specific promoter. 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 astroglia cells for the genetic control of exocytosis (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 astroglia cells,

genetically encoded calcium indicator (GKKI) (GCaMP3 and others) as a marker gene,

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

The genetic construct for transfection of astroglya cells LVV-GFAP-GCaMP3 was constructed on the basis of an improved lentiviral shuttle vector pTYF-SW-Linker containing recognition sites for several restriction endonucleases (SceI, EcoRV, NheI, SalI, SmaI, BamHI, S , SpeI, MluI, XhoI, NotI, ClaI, I-SceI, KpnI), HIV-1 Rev-sensitive element, providing Rev-dependent export of mRNA from the nucleus to the cytoplasm, CMV-TATA-TAR - recombinant immediate-early promoter / enhancer cytomegalovirus with substitution in the 5'-U3 long terminal repeat, gag - viral protein th nucleus, cPPT-FLAP - the sequence of the central polypurine tract and polyadenylation site.

To increase the titer of the vector and improve the expression of the transgene into the structure of the shuttle vector pTYF-SW-Linker, previously hydrated at NotI and ClaI sites, an amplified fragment of the marmot hepatitis virus post-transcriptional regulatory element (WPRE) was cloned - the sequence SEQ ID N1. For this, we carried out a polymerase chain reaction with specifically selected primers WPRE_Forw (CCGCTCCCGTTATTT) WPRE_Rev (TTTATTGCCCTCGCC). The resulting plasmid was produced in preparative quantities for subsequent cloning into it of the GCCI.

Using a pair of primers GCaMP3_Forw (AGAGCTGGTTTAGTGA) and CCaMP3_Rev (AATGTGGTATGGCTG), using the plasmid pN1-Lck-GCaMP3 (Clontech) as a template, the PCR product of 1570 bp was amplified. - the sequence of SEQ ID N2, which was cloned pTYF-SW-Linker-WPRE, previously hydrolyzed at the sites Bg1II and NotI. The resulting plasmid was produced in preparative quantities for subsequent cloning into it of a cell-specific promoter.

For specific transfection of brain astroglia cells, the cell-specific glial fibrillar acid protein (GFAP) promoter was cloned into the sequence of the genetic construct. 704 bp PCR product - the sequence of SEQ ID N3 was obtained by amplification using a pair of primers GFAP_Forw (GACGCTGCTCTGACA) and GFAP_Rev (CTGAGACTGGGGAAT) using the plasmid pGfaABC1D-nLac as a template. The nucleotide sequence fragment thus obtained was cloned into pTYF-SW-Linker-WPRE-GCaMP3, previously hydrolyzed at the MluI and SpeI sites.

Thus, we obtained SEQ ID N4 genetic construct - LVV-GFAP-GCaMP3 for transfection of astroglia cells and expression of GCa GCaMP3 in them.

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

The claimed method for the genetic control of exocytosis based on genetic constructs is carried out by transfecting cultures of primary astroglia cells obtained from three-day old rat pups and grown on Dulbecco's modified Eagle medium (DMEM) (with 4.5 g / l glucose, glutamine, and pyruvate) with the addition of 10% fetal bovine serum. At the same time, the day before transfection, the cells are seeded in 24-well culture plates with a cell density of 5 × 10 ⁴ per well and incubated for 8-14 hours, after which the astroglia cells are transfected with the developed genetic construct -SEQ ID N4 - LVV-GFAP-GCaMP3 in the presence of polybrene (8 μg / ml), washed with phosphate buffer and cultured for 40-52 hours in DMEM. After that, exocytosis was controlled by inducing cascades of P2X and P2Y purinoreceptors, β-adrenergic receptors. Coverslips with cells are washed from DMEM growth medium with Hanks complete balanced solution (HBSS) at room temperature and transferred to the experimental cell. The cells are left alone for 30 minutes at room temperature. The experimental cell is transferred to the stage of a laser scanning confocal microscope and the settings for the fluorescence exciting laser and light filters for recording fluorescence are set. To activate P2Y dependent Ca ²⁺ cascades, 10 μM 2-MeSATP is added to the experimental cell and a change in the fluorescence level is recorded. To activate P2X-dependent Ca ²⁺ cascades, 10 μM a, b-MATP is added to the experimental cell and a change in the fluorescence level is recorded. To activate the p-adrenergic dependent Ca ²⁺ cascades, 50 μM isoproterenol is added to the experimental cell and a change in the fluorescence level is recorded.

The use of the claimed invention allows genetic control of exocytosis based on genetic constructs (for transfection of astroglia cells (SEQ ID N4-LVV-GFAP-GCaMP3), since the genetically encoded calcium indicator GCaMP3 is built into the developed genetic construct, which allows controlling astroglia characteristic of cells Ca2 ⁺ - dependent exocytosis Furthermore, the use of the claimed invention allows the in vitro visualization of transfected cells by embedding in develop. genetic construct genetically encoded calcium indicator.

Claims (1)

The genetic construct of SEQ ID N4 for implementing the method of genetic control of exocytosis by transfection of astroglia cells with the ability to carry out targeted control of intracellular cascades associated with astroglia vesicular transport and also to express a genetically encoded calcium indicator in them, which provides genetic control of exocytosis, in which of the vector, an HIV-1 based lentiviral vector modified with the sequences of SEQ ID N1 encoding marmot hepatitis virus st-transcriptional regulatory element (WPRE), SEQ ID N2 encoding a genetically encoded calcium indicator (GCaMP3), SEQ ID N3 encoding a cell-specific promoter of glial fibrillar acid protein (GFAP), the expression of which leads to cell-specific expression genetically encoded calcium indicator in brain astroglia cells.

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