Method of providing safety of application of pluripotent stem cells in tissue-substituting therapy by means of artificial chromosomes
SUBSTANCE: invention relates to field of medicine, genetic engineering and biotechnology. Claimed is method of providing tumour-free tissue-substituting therapy on the basis of obtained from adult somatic cells induced pluripotent stem cells (iPSC), where the latter are genetically modified by means of artificial chromosome (AC), carrying bicistronic cassette with suicide-gene and gene of sensitivity to antibiotic under control of regulatory element, specific for pluripotent stem cells, with modified iPSC being selected in presence of respective antibiotic, absence of AC integration into iPSC genome is confirmed, are introduced into recipient organism directly, without preliminarily differentiation in vitro, after 1-14 days patients are given a 5-10-day course of therapy with inductor of toxicity of suicide-gene product.
EFFECT: invention makes it possible to reduce to zero risk of cancer transformation of transplanted cells and development of teratomas, increase clinical efficiency of recovery of cell mass of injured organ or tissue, reduce waiting time for potential recipients.
6 cl, 1 tbl, 1 dwg
The development of effective and safe approach to transamidase based on a combination of technologies pluripotent stem cells, genetic sensitization and technology artificial chromosomes (THEM).
Cultured pluripotent stem cells, which primarily are derived from somatic cells by forced expression of 4 transcription factors (Oct4, Sox2, Klf4, yssc) induced pluripotent stem cells (ipscs), have been intensively studied recently due to two unique properties of these cells-the ability indefinitely to share ex vivo without changing its properties, as well as the ability to differentiate into all known cell types of the body. ipscs cells are the most versatile source for denisemasino, and it is important to note that can be obtained in their somatic cells of the patient, i.e., they are fully immunologically compatible. Unsolved problem on the way to the use of ipscs cells in clinical practice, is tumorogenity - an inherent property of these cells. Thus, in mice it was shown that the introduction of cells to the recipients can result in a risk of teratoma formation is a fast - growing benign tumors containing a variety of cell types of all three germ layers. � the other hand, existing methods of directed differentiation of ipscs in vitro provide the most heterogeneous population in which virtually always present residual undifferentiated ipscs that can, in the transplants to give rise to teratoma formation.
Develop one or more ways to ensure the safe use of ipscs is in the focus of research in many laboratories in Russia and abroad. Currently proposed methods are ineffective and do not provide an adequate level of security, thereby depriving laid in pluripotent cells is of great practical potential and significantly hinders the prospect of using these unique cells in clinical practice. Recently, we proposed a new approach to solve laboratory ipscs, which is based on genetic sensitization (RF patent No. 2009143025). This approach minimizes the risk of teratoma formation, originating from donor pluripotent stem cells at the injection site or in any of the distal site in the recipient. This is achieved sensitization ipscs through the stable introduction into the genome of the host cell suicidal DNA-cassettes, predstavljajushej a gene-suicide (thymidine kinase or TK) under the control of the regulatory region of Oct4, providing Express�Yu this cassette only in undifferentiated ipscs, but not descended from them differentiated cells. Such selectivity expression cassette allows you to eliminate (using ganciclovir or GCV) or in culture, or even directly in the recipient's body bearing its teratogenic residual ESCs and ipscs cells, without affecting the differentiated cells. Delivery of the specified DNA cassettes into ipscs carried out with this method by electroporation of plasmid vectors, or by infection of these cells by viral vectors carrying a gene-suicide. Despite the importance of the proposed approach, its disadvantages were (1) epigenetic instability suicidal cassette in transgenic or proviral DNA in the genome, with the resultant risk of loss of control over the formation and growth of teratoma formation and (2) the likelihood of dysfunction of host genes, for example encoding tumor suppressors, while random integration into the genome (insertional mutagenesis), leading to malignant transformation of differentiated descendants ipscs. Thus, on the one hand, for effective sensitization requires stable integration into the genome, and such integration is highly undesirable due to the high risk of insertional mutagenesis. Thus, for needs improvement by increasing security �of thoda genetic sensitization there is an urgent need in geintegreerde in hozyaiskoi genome and stable delivery systems and the maintenance of foreign DNA.
Geintegreerde vector systems such as adeno-associated viruses, which do not allow to solve this problem, because adenoviruses are not stably maintained in the cells and are lost after several cell divisions.
Perfect geintegreerde stable vector system are artificial chromosomes (THEM). Proposed in the present application is an approach that combines several modern cellular and genomic technologies, allows to solve the above-described problem in an original way. Administered and maintained suicidal cassette in the host cell would not be in the form of a stably integrated transgene, or provirus, and in THEIR composition, which does not disturb the integrity of the genome and are not susceptible to epigenetic damping.
Technology (from the English. Human Artificial Chromosomes or US) is a fast - growing lately area in post-genomic biology, which is primarily designed for studying the molecular mechanisms of cancer, but may be of great interest in the study of stem cells and gene therapy. A distinctive feature of THEM is their ability to exist in the nucleus in the form of individual chromosomes that do not violate the integrity of the host genome, its huge capacity, mitotic and transcriptional stability. This technologist�I got its further development, when the proposed method of controlled destabilization and removal from the cell through the introduction of specific sites, tetO, prevent the binding of kinetochores in centromere sequences by landing on them doxycycline-reguliruemykh of repressors, such as tT-KRAB (Ebersole et al., 2005; Kouprina et al., 2003; Nakano et al., 2008; Okada et al., 2007).
Creation method used in the present invention of THEIR class, created on the basis of altenach replays centromeres human DNA, described in patents EP 1866442, WO /2008/013067, EP 2060633. These patents, however, do not consider THEM as a carrier of the gene-of samoubiicy to eliminate ESCs or ipscs or for any other purposes.
As a prototype method of genetic sensitization we have chosen the method described in patent US 6576464. At the first stage in undifferentiated stem cells is administered a nucleic acid molecule consisting of 2 elements. The first element is a nucleic acid sequence encoding a product that is lethal to the cells in which it is expressed, or make a cell in which it is expressed susceptible to the lethal action of external factors. The second element is the element of control of transcription, which provides the expression of the first element predominantly in undifferentiated stem cells. The first element soduritena, encoding a toxin or a protein inducing cell death (suicide genes), such as an enzyme (TK) converts the drug substrate (widely used antiviral drug ganciclovir or GCV) into the substrate, lethal for the cell. The expression encoding an enzyme genes is under the control of the promoter (endogenous transcriptional control element) that is specific to undifferentiated stem cells (for example, an enhancer of the Oct4 gene (AV), made with a minimal promoter of herpes virus (tk) is designated in the application as 2A2Btk). In the second stage, carry out the induction of directed differentiation of stem cells: the parameters of this phase depend on the objectives of the Protocol (the direction of cell differentiation). In the third phase in cultured cell population add ganciclovir, which selectively metabolized undifferentiated stem cells (expressing genes Oct4) and induces in these cells, cell death (apoptosis). This provides selective ablation of residual undifferentiated cells from a heterogeneous cell population. According to the authors of the invention, the proportion of remaining undifferentiated cells does not exceed 0.02% of total cells in the population (US 6576464). Meanwhile, even a small number of undifferentiated cells, persistent in Goethe�ogen the population can cause the development of teratomas in the site/sites of cell transplantation. In addition, the patent - prototype (US 6576464), does not offer any way of increasing the efficiency of technologies for directed differentiation. Third, the patent-prototype relies on transgenic delivery method, fraught with insertional mutagenesis and risk of silencing gene expression-suicide, with the resultant loss of control of teratogenesis.
The aim of the present invention is to enable effective chinesemedicine therapy using ipscs in nullifying the risk of teratoma formation, originating from these cells at the injection site or in any of the distal site in the recipient, and when the reduction to zero of the risk of insertional mutagenesis and epigenetic silencing of suicidal cassettes. This is achieved in this application by introducing into the genome ipscs derived from somatic cells of adult organisms in a common manner, and in maintaining a stable condition of multicomponent genetic constructs (e.g. bicistronic magazine) in THEIR composition. This cassette contains the gene for suicide, such as the TC, under the control of the regulatory element-specific ipscs, such as the enhancer/promoter of the Oct4 gene. For positive selection cassette further comprises a resistance gene to the antibiotic, for example puromycin under to�control of the same regulatory element. After the transfer, containing a suicidal cassette from the donor cells, in which it was collected (e.g., Cho cells) in ipscs method of microdelay or by any other method, the cell population is treated with an antibiotic, such as puromycin, for positive selection of clones of ipscs, included in its composition. After this, the cells are administered, without any pre-differentiation in culture, directly into the bloodstream, organ, or tissue recipients. Through 1-14 days, the recipient is receiving a course of therapy with ganciclovir.
The positive effect of the application of the present invention is: (1) the reduction to zero risk in recipients of teratoma formation after transplantation of the cell material, 2) the reduction to zero of the risk of cancerous degeneration of the transplanted cells by insertional mutagenesis, 3) to increase the clinical effectiveness of replenishment of damaged cell mass of an organ or tissue, (4) reduction, due to the absence of the stage of differentiation in culture, the duration of the waiting period for potential recipients.
The novelty of the present invention is that undifferentiated ipscs bearing described above gene-suicide (e.g., TC), and optionally, a marker for positive selection of cells (e.g., puromycin) is injected, without any Lieb� pre-differentiation in culture, in krautok, organ, or tissue recipients to fill cell mass or to correct paracrine, contractility, neurotransmitter or other physiological functions. After a certain incubation period (1-14 days), during which the ipscs colonise the damaged organ, tissue, and independently differentiate into the required cell type under the influence of the microenvironment, the recipient is entered orally, intramuscularly or intravenously gene corresponding to the type of suicide inductor (in the case of TC, applies known antiviral drug ganciclovir). Gene expression of suicide in all residual undifferentiated ipscs (provided that pre-breeding for resistance to puromycin) makes these cells sensitive to ganciclovir, leading to their apoptotic death, thereby preventing Vozniknovenie of teratoma formation or stop their growth in the early stages.
The invention may find an important application in the treatment of diseases such as diabetes mellitus (by replenishing the pool of insulin-secretaryship beta cells of the pancreas), liver failure (replenishing pool of hepatocytes), consequences of heart attacks (recovery and vascularization of the heart muscle), neurodegenerative diseases and spinal damage�mation (recovery of neuronal tissue), syndrome Duchesne (recovery of musculoskeletal muscles), etc.
The efficiency of the developed approach for genetic sensitization on the basis of THEIR approach is illustrated by the following example. We created earlier in vitro altenau THEIR bearing bicistronic cassette 2A2Btk-TKiresPuro (further still, as shown in figure 1) was transferred into ipscs mouse cells by the method MST (microcell-also been other ideas where chromosome transfer). Logarithmically growing (50-70% plantnet) so Cho cells (12x bottles of 25 cm2, centrifuged (8000 rpm, 37°C, 60 min) in medium containing 10 μg/ml of cytochalasin V. the Precipitate was resuspended in 2 ml of warm DMEM medium without serum and incubated on ice for 20 min, Then a suspension of microdelay sequentially filtered through 8, 5 and 3-mm Swinnex filters (Whatman), centrifuged at 2000 rpm, 5 min at room temperature and RNA was resuspended in R buffer (Wako). Microsemi were incubated with 105ipscs derived from mouse fibroblasts using lentiviruses, for 15 min at 37°C. the cells then were added PEG-1000 and left for 30 s. After that, cells were washed in medium, and the next day were scattered at low density and selected in the presence of 2 μg/ml puromycin. Through 4 days after the merger-passaged cells on 10-cm culture dishes (Falcon, USA), previously coated with a monolayer of mitotically inactivated MEF, and were cultured in with one of�next environments: 1) N2B27, a mixture of penicillin and streptomycin (Gibco, USA), 2 mm L-glutamine, 0.1 mm β-mercaptoethanol (Gibco, USA), GSK3p inhibitor - 3 µm CHIR99021 (Stemgent, USA), an inhibitor of MEK - 0.5 μm PD0325901 (Stemgent, USA) and LIF (leukemia inhibitory factor) 1000 U/ml (PAA, Austria) (Buehr et al., 2008) (medium (N2B27+2i+LIF)); 2) medium-Knockout-DMEM (Invitrogen, USA) as the basis, containing 20% serum substitute KnockOut-SR™ (Invitrogen, USA), a mixture of penicillin and streptomycin, 2 mm L-glutamine, 0.1 mm β-mercaptoethanol, 1% nonessential amino acids (Gibco, USA), 3 μm CHIR99021, 0.5 μm PD0325901, an inhibitor of ALK5 - 0.5 μm A-83-01 (Tocris Bioscience, USA) and LIF 1000 U/ml (Li et al., 2009) (environment(KSM+3i+LIF)). Through 1012 d after subculturing colonies of ipscs were individually selected, treated with 0.25% trypsin (TrypLE™ Express, Gibco, USA) and transferred to wells of 96-well plates, previously coated with mitotically inactivated MEF, and continue to cultivate for 1-2 passages in the respective environments. The efficiency of the transfer of macrocera was 0.023%, (23 clone 105source ipscs). In the future, the cultivation of all cultures obtained ipscs carried out in a medium (N2B27+2i+LIF). Replacement of the medium produce every 1-2 days. Next 6 of 23 source derived ipscs clones, most morphologically relevant undifferentiated ipscs were analyzed using FISH analysis using samples against altenach repetitions of alphoid centromere-and THEIR gene thymidine kinase (TK). FISH-analysiscanal, in all of the 6 selected ipscs clones (1), (2) maintained a normal diploid karyotype.For preliminary health evaluation so approach, undifferentiated so-ipscs cells (two different clone) was injected under the skin of immunodeficient mice lines NUDE. 10 days later the mice underwent a 7-day course of injections of GCV (10 mg/kg body weight), and after another month, evaluated the presence at the injection site of teratoma formation. The result of the experiment is shown in Table. It is seen that the course of injections completely suppresses the formation of teratoma formation from now. At the same time, the introduction of GCV mice, the growth of teratoma formation from control ipscs, which directly pointed to the essential role of suicide-THEIR control over this process. After a further 5 weeks (a total of 9 weeks after injection of cells against 8 weeks in TK) mice further examined, which confirmed the previous conclusion. We conducted a 10-day course of injections of GCV that was sufficient to inhibit teratoma formation.
Thus, the presence ipscs cells so ensure the death of these cells when exposed to GCV, thereby suppressing the formation of teratoma formation. In control mice after the introduction of the so-ipscs that have not undergone a course of GCV, teratomas developed with 100% frequency. Cassette 2A2Btk-TKiresPuro in the composition so gene expresses suicidal herpes virus, thymidine kinase (T�), product which converts GCV (analog dezoksiguanozina) in fosforilirovannoyj form GCV which, once incorporated into DNA, competitively inhibits DNA polymerase. This leads to suppression of DNA synthesis by inhibiting chain elongation of DNA and cell death. In the proposed system TC is used for the selective destruction of residual undifferentiated ipscs, but not differentiated derivatives due to the fact that this gene placed under the control of an enhancer of the Oct4 gene, which is active only in the first cell types.
Another control mouse was injected with ipscs wild type, were followed by a course of GCV. Last, as expected, had no antitumor effect in consequence of the fact that the cells were not present TK gene necessary to make these cells sensitivity to GCV (Table. 1).
The list of references
1) Ebersole, T., Okamoto, Y., Noskov, V. N., Kouprina, Ν., Kim, J. H., Leem, S. H., Barrett, J. C., Masumoto, H., and Larionov, V. (2005). Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation. Nucl Acids Res 33, e130.
2) Kouprina, N., Ebersole, T., Koriabine, M., Pak, E., Rogozin, I. B., Katoh, M., Oshimura, M., Ogi, K., Peredelchuk, M., Solomon, G., et al. (2003). Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes. Nucl Acids Res 31, 922-934.
3) Nakano, M., Cardinale, S., Noskov, V. N., Gassmann, R., Vagnarelli, P., Kandels-Lewis, S., Larionov, V., Earnshaw, W. C., and Masumoto, H. (2008). Inactivation II of a human kinetochore by specific targetng of chromatin modifiers. Dev Cell 14, 507-522.
4) Okada, T., Ohzeki, J., Nakano, M., Yoda, K., Brinkley, W. R., Larionov, V. and Masumoto, H. (2007). CENP-B controls centromere formation depending on the chromatin context. Cell 131, 1287-1300.
1. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells to induced pluripotent stem cells (ipscs), characterized in that the last genetically modified by means of artificial chromosome (THEM), carrier bicistronic the cassette with the gene-suicide genome and sensitivity to the antibiotic under the control of the regulatory element specific for pluripotent stem cells, wherein the modified ipscs are selected in the presence of the corresponding antibiotic, confirm the lack of integration in the genome of ipscs, injected into the body of the recipient directly, without prior in vitro differentiation, after 1 to 14 days recipients spend 5-10-day course of therapy inducer of toxicity of the gene product-suicide.
2. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells to induced pluripotent stem cells (ipscs) according to claim 1, characterized in that the antibiotic take puromycin.
3. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells induced pluripo�/ stem cells (ipscs) according to claim 1, characterized in that the gene-suicides take TK (thymidine kinase) and as an inducer of toxicity GCV (ganciclovir).
4. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells to induced pluripotent stem cells (ipscs) according to claim 1, characterized in that the gene-suicides take TDR (the receptor for diphtheria toxin), and as an inducer of toxicity of diphtheria toxin.
5. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells to induced pluripotent stem cells (ipscs) according to claim 1, characterized in that as a regulatory element specific for pluripotent stem cells take enhancer/promoter of the Oct4 gene.
6. The way to ensure basophilia chinesemedicine based therapies derived from adult somatic cells to induced pluripotent stem cells (ipscs) according to claim 1, characterized in that for genetic modification using THEM created on the basis of altenach retry centromeric regions of human chromosomes.
SUBSTANCE: claimed invention relates to the field of biotechnology, namely to the preliminary estimation of the efficiency of the autologic cell material transplantation to stimulate the growth of blood vessels, and can be applied in medicine. Claimed is a method of the complex estimation of the angiogenic potential of progenitor cells in patients with cardiovascular diseases, tested on mesenchymal stromal cells of the adipose tissue (MSC-AT) of patients with ischemic heart disease and including the measurement of content of mRNA and proteins of basic angiogenic factors, produced by the progenitor cells such as the vascular endothelial growth factor (VEGF), the placental growth factor (PIGF), the hepatocyte growth factor (HGF), angiopoetin-1 and angiogenin, the angiogenic activity of total cell secretion products, as well as the estimation of the ability of the progenitor cells to stimulate the vascularisation of subcutaneous Matrigel implants, introduced to immunodeficient mice. As the screening method used is a simpler and more available but less informative method of express-assessment of the angiogenic properties of the progenitor cells, based on the measurement of the angiogenic activity of the total cell secretion products.
EFFECT: invention makes it possible to carry out testing of the autologic cell material obtained from the patients, including those with ischemic heart disease, before transplantation in order to choose the optimal tactics of cell therapy aimed at the stimulation of the growth of blood vessels.
2 cl, 2 dwg, 4 tbl, 4 ex
SUBSTANCE: invention refers to biotechnology. A method provides preparing a suspension of pluripotent stem cells and adding a compound able to inhibit Rho-kinase activity to the suspension. That is followed by adding the cell suspension to a flat carrier, and the compound is removed after the cells has been fixed.
EFFECT: what is described is the method for human pluripotent stem cell fixation on the flat carrier.
14 cl, 32 dwg, 10 tbl, 14 ex
SUBSTANCE: method of detection of intracellular proteins using fluorescein-5-isothiocyanate (FITC) in various types of mammalian cells is provided, which are characterised by different levels of the synthetic process, using the method of confocal laser microscopy. The mammalian cells are fixed by paraformaldehyde (PFA) preventing the extraction of proteins in subsequent stages of staining, permeabilised with detergent, then stained for 2 h with FITC in a concentration of 1 mcg/ml. The cells are enclosed in Mowiol 4-88 adding DABCO (1,4-diazabicyclo[2.2.2] octane). The resulting preparations are used to analyse the localisation and the content of the protein component of the cells by confocal microscopy.
EFFECT: invention enables to obtain information and to investigate the location of proteins in the cells and to carry out a comparative analysis of protein content in cells with different levels of metabolism.
7 dwg, 7 ex
SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.
EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.
28 cl, 4 dwg, 7 tbl, 10 ex
SUBSTANCE: group of inventions refers to medicine, namely to orthopaedics, and can be used for producing a transplant for long bone tissue repair. That is ensured by collecting bone marrow aspirate into lithium heparin test tubes, diluting with phosphate salt buffer, filtered through a filter with pore size 70 mcm and centrifuging for 10 min at 400 g. That is followed by inoculating flasks 150 cm2 with the produced mesenchymal stem cells (MSCs) at 1×106 cells/cm2, culturing at temperature 37°C and 5% CO2 in air, in the Dulbecco's Modified Eagle's medium containing glucose 1 g/l and less, with 10% embryo calve serum. The medium is changed every 3 days. Before preparing the transplant, the cells are removed from the substrate with 0.05% trypsin EDTA, washed and re-suspended. The cell suspension is applied on the matrix, incubated for 3 hours at temperature 37°C with rocking in a rotation shaker at 25 rpm. The matrix is filled with the DMEM medium with 10% bovine foetal serum, dexamethasone 10-7 M, β-glycerophosphate 10 mM, ascorbate-2-phosphate 0.05 mM, 1% streptomycin 100 mcg/ml or penicillin 100 units/ml. The MSCs are cultured for 28 days. What is also presented is a method for long bone tissue repair.
EFFECT: group of inventions provides the uniform bone tissue repair in replacing the bone tissue defect of critical size with the transplant.
3 cl, 2 tbl, 6 ex
FIELD: veterinary medicine.
SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.
EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.
1 tbl, 3 ex
SUBSTANCE: invention refers to methods for cell and tissue protection against a hypoxic injury and can be used for developing remedies for hypoxic and ischemic injuries. The developed method for protection is based on cell treatment by substances increasing a level of glutathionylation of Na,K-adenosine triphosphatase catalytic subunit that reduces to its activation. The obtained results can be actual for biological research institutions, medical facilities, particularly cardiologic clinics, as well as biotechnological companies for transplantology and tissue engineering.
EFFECT: presented method can be used for research activities aiming at creating the therapeutic agents for relieving tissue injuries, particularly myocardial tissue in hypoxia and hypoxia/reoxygenation.
SUBSTANCE: invention refers to a method for developing Alzheimer disease cell models for testing medical effectiveness of chemical substances to be further used in medicine, more specifically in treating individual's neurodegenerative diseases caused by neurotoxic effects of beta-amyloid aggregates. A molecular cytotoxic agent is a synthetic analogue of human beta-amyloid 1-42 isomerised in asparagic acid residue in position 7 ([isoD]) (amino acid sequence: DAEFRH[isoD]SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). The additional advantage of the present invention is applicability of primary cell cultures as Alzheimer disease models.
EFFECT: advantage of these Alzheimer disease cell models as compared to currently used exogenous-induced models, wherein the cytotoxic effects are caused by a non-modified form of beta-amyloid is a higher ability of the isomerised beta-amyloid to induce both cell necrosis and apoptosis.
SUBSTANCE: invention refers to biotechnology and concerns a method for producing progenitor cells from mammalian myocardial samples. A presented method involves milling the samples, implanting them on culture dishes coated so that to provide adhesion; culturing the samples in the hypoxic environment so that to provide the sample adhesion to form a cell culture; recovering cells from the cell culture by enzymatic treatment; culturing the recovered cells until forming cell aggregates, and processing the cell aggregates with an enzyme solution to produce the progenitor cells of myocardium.
EFFECT: presented invention can be used in medicine and veterinary science for studying the myocardium regeneration mechanisms and stem cell biology, as well as for treating cardiac diseases.
10 cl, 2 dwg, 2 ex
SUBSTANCE: invention refers to biotechnology and medicine. What is presented is the mus musculus's hybrid culture cell strain α-producing monoclonal antibodies specific to human granulocyte colony-stimulating factor (GCSF) deposited in Special Cell Culture Collection of Institute of Cytology of the Russian Academy of Sciences, under No. PKKK ("П") 662 "Д".
EFFECT: invention enables extending the range of strains producing GCSF-specific monoclonal antibodies used for research and medical applications.
2 dwg, 2 tbl, 2 ex
SUBSTANCE: as the present invention the method for production of polyfunctional magnetic nanoparticles based on bacterial magnetosomes and the hybrid protein MGG is provided, which enables to obtain magnetosomes binding immunoglobulins of the IgG class on the fragment Fc. The result is achieved in that in the lipid membrane of bacterial magnetosomes by ultrasonic treatment the hybrid protein MGG is integrated, which amino acid sequence comprises the transmembrane domain and the binding area of immunoglobulins.
EFFECT: obtaining polyfunctional magnetic sorbent bearing on the surface of magnetic nanoparticles of ligand, that enable to connect to the particles the immunoglobulins of IgG class.
SUBSTANCE: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.
EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.
3 cl, 7 dwg, 4 ex
SUBSTANCE: invention refers to biotechnology, more specifically to a method for introducing an siRNA molecule into the cytosol of a cell, and can be used in medicine. The method involves contacting said cell with an siRNA molecule, a carrier and a photosensiting agent, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. The carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, branched polyethyleneimine (PEI), a betacyclodextrin amine polymer, a PAMAM dendrimer molecule, and a cationic peptide such as polyarginine or L- or D-arginine copolymers. The method is used to inhibit the target gene expression, to obtain a cell or a cell population containing the siRNA molecules, as well as to treat or to prevent a disease where the inhibition of one or more genes may be effective, including to treat a malignant growth.
EFFECT: invention enables the PCI-mediated site-specific siRNA delivery into the cytosol of a cell.
27 cl, 15 dwg, 1 tbl, 13 ex
SUBSTANCE: immunostimulatory complex is described, which includes an RNA in the form of a complex with one or more oligonucleotides. Besides, the oligonucleotide has properties of peptide penetrating into a cell (CPP), contains from 8 to 15 amino acid remains and is characterised by the following general formula: (Arg)1(Lys)m(His)n(Orn)o(Xaa)x. At the same time the main number of remains is selected from Arg, Lys, His, Orn.
EFFECT: invention may be used for transfection of a cell, tissue or organism or for modulation, preferably induction or increasing immune response.
16 cl, 22 dwg, 11 ex
SUBSTANCE: method includes processing of plant cells with intact cellular barriers by complex of carrier - carried material consisting of at least one carried polypeptide and/or polynucleotide component connected with the carrier - internalised positively charged peptide (IPCP), and designed for obtaining the transduced cells and propagation of plant cells for cultivation of plant. And these plant cells are somatic cells preliminary treated with agent permeabilising cells, or gametophytes.
EFFECT: invention enables to obtain effectively genetically engineered plants under conditions of intense cellular uptake of the complex of carrier-carried material.
13 cl, 11 dwg, 5 tbl, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of molecular biology and biotechnology and can be used in medicine and in pharmaceutical industry. RNA molecule, capable of target-specific RNA interference, represents double-stranded RNA molecule, 23 nucleotides long, which has 3'-overhang from 1-5 nucleotides. It is obtained by joining two RNA strands and used for obtaining pharmaceutical composition. When introduced into multicellular eukaryotic organism or in a cell of multicellular eukaryotic organism, said RNA molecule ensures promotion of target-specific RNA interference and leads to reduction of target-gene expression level or to target-gene knockout. Method of promoting target-specific RNA interference by means of said RNA molecule is applied for determination or modulation of gene function. Cell, containing endogenic target nucleic acid, RNA molecule, capable of target-specific RNA interference, and exogenic target nucleic acid, is used in analytical procedures.
EFFECT: application of invention ensures target-gene silencing, mediated by target-specific RNA interference.
40 cl, 23 dwg, 3 ex
SUBSTANCE: method provides contact of said cell with a peptide nucleic acid (PNA) molecule and a photosensitising agent and cell exposure to light at wave length effective to activate the photosensitising agent where said PNA molecule is conjugated with positive peptide. Also, there are described compositions containing such conjugated PNA molecules, the cells produced with the use of the method, and application of the method.
EFFECT: effective cell capture of peptide nucleic acid molecules conjugated with positive peptide.
37 cl, 13 dwg, 9 ex
SUBSTANCE: invention refers to biotechnology and genetic engineering, namely to a genetically engineered construct pGoatcasGCSF for human granulocyte colony-stimulating factor (GCSF) expression . The offered invention can be used for producing transgenic animals that are human granulocyte colony-stimulating factor producers. It involves creating the genetically engineered construct pGoatcasGCSF of the size 6386 bps, and a specified nucleotide sequence shown in SEQ ID 1. The genetically engineered construct pGoatcasGCSF includes a 5'-regulatory sequence of the goat CSN1S1 asi-casein gene of the size 3387 bps connected with the full-size human GCSF gene of the size 1485 bps, and a 3'-flanking region of the cow gene CSN1S1 of the size 1514 bps.
EFFECT: stable effective level of human GCSF expression in milk of the transgenic animals, eliminated possibility of ectopic transgenic expression.
9 dwg, 2 tbl, 6 ex
SUBSTANCE: invention relates to carbosilane dendrimers and synthesis method and use thereof. Disclosed are novel dendrimers, the ends of branches of which include primary, secondary, tertiary and quaternary amino groups, synthesis method thereof, composition based on said dendrimers and versions of using said dendrimers. The disclosed dendrimers may be used to transport anionic molecules in blood, such as nucleic acid molecules, including ODN and RNAi molecules and other anionic medicinal agents with which they can interact, thus protecting them from interaction with proteins in the plasma and/or enhancing their degree of penetration into target cells. The dendrimers can be used to bind anionic molecules with surfaces, and can also be administered as active components for preventing or treating diseases caused by viruses such as HIV or hepatitis C, or prions whose life cycle can be disrupted by dendrimers.
EFFECT: obtaining novel carbosilane dendrimers, having a wide range of application in medicine and which are processable and biocompatible.
172 cl, 35 dwg, 19 tbl, 57 ex
SUBSTANCE: invention relates to a novel chemical compound, specifically to rac-N-[2,3-di(tetradecyloxy)prop-1-yl]pyridinium bromide, which can deliver nucleic acids into mammal cells: .
EFFECT: compound has low toxicity and in form of an alcohol solution, the compound can deliver nucleic acids into mammal cells.
1 cl, 5 ex, 4 tbl, 1 dwg
SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.
EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.
5 cl, 1 tbl, 8 dwg, 4 ex