RU2459870C2 - Promoter for tissue-specific gene expression in terminal mammal tissues - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: presented promoter consists of a sequence of the length of 3574 base pairs above an initial point of the human transcription gene NDUFV1 with a remote sequence of the length of 91 base pairs immediately above the initial point of said gene transcription.

EFFECT: invention provides creating an artificial strong promoter which may be applicable for the therapy of terminal tissues, producing transgenic animals or modified cell lines with the desired functions.

5 dwg, 1 tbl, 4 ex

Description

The present invention relates to biotechnology, in particular to animal genetic engineering, to artificial DNA constructs demonstrating the high activity of the transcriptional promoter from which the expression of an arbitrary nucleotide sequence transcribed by RNA polymerase II is carried out. The activity of the promoter is limited to germ cells (HA) and the corresponding mammalian tissues. The promoter can be used to treat terminal tissues, as well as to create transgenic animals or modified cell lines with specified functions.

Gene expression is provided by genomic regulatory elements, including, but not limited to, promoters, enhancers, and silencers of various structures.

These regulatory elements contain a unique set of shorter motifs that bind regulatory proteins, for example, various transcription factor proteins. The resulting unique repertoire of regulatory protein binding sites, apparently, mainly determines the transcriptional control of genes. Some genes are transcribed in all tissues of the body, some in several tissues, and some others only in a single tissue. Regulatory regions of genes characterized by such tissue-specific transcription can be used to create genetic engineering constructs for the specific expression of sequences of interest (RNA or protein) in a strictly defined tissue (or group of tissues) of the body. Such a task can be posed, for example, when creating a gene therapy strategy or when solving research problems. Gene-therapeutic applications can be aimed at filling the deficiency of any protein product in the body (for example, introducing into the hematopoietic stem cells of patients suffering from hereditary forms of hemophilia structures containing an expression cassette with a normal copy of the coagulation factor IX gene (Mates, L., Chuah, MK, Belay, E., Jerchow, B., Manoj, N., Acosta-Sanchez, A., Grzela, D.P., Schmitt, A., Becker, K., Matrai, J., Ma, L., Samara-Kuko, E., Gysemans, C., Pryputniewicz, D., Miskey, C., Fletcher, B., Vandendriessche, T., Ivies, Z., and Izsvak, Z, (2009). Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41, 753-61)), as well as the fight against autoimmune and cancer diseases (de Bruin, E.C. and Medema, JP (2008). Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer Treat Rev 34, 737-49).

Currently, intensive development of new generation drugs is underway that can selectively affect cancer cells with a reduced amplitude of side effects on normal body tissues. The principle of action of such drugs is based on knowledge of the nature of the development of a particular type of tumor and the biochemical processes that occur in them, often taking into account the individual characteristics of the patient.

Known drugs that specifically recognize and kill cells expressing certain surface antigens (often cell growth factor receptors), cells with inactivated cancer suppressor genes, cells with aberrantly blocked apoptosis pathways (Frenzel, A., Grespi, F., Chmelewskij, W., and Villunger, A. (2009). Bcl2 family proteins in carcinogenesis and the treatment of cancer. Apoptosis 14, 584-96).

Liposomes, viral particles, polyplexes (Sobolev, AS (2008). Modular transporters for subcellular cell-specific targeting of anti-tumor drugs. Bioessays 30,278-87), naked DNA or DNA liposaccharide complexes are used as delivery vehicles for gene therapy drugs. (Yu, H., and Wagner, E. (2009). Bioresponsive polymers for nonviral gene delivery. Curr Opin Mol Ther 11, 165-78).

This approach involves the introduction of genetic engineering constructs into the cells of the body that specifically affect cancer cells and lead to the death of the latter. Widely considered, for example, are the prospects for specific activation of intracellular oncosuppressive genes (Garcia-Echeverria, C., and Sellers, W.R. (2008). Drug discovery approaches targeting the PI3K / Akt pathway in cancer. Oncogene 27, 5511-26). A very promising approach seems to be delivery of killer genes (or “killer genes”) to the cell under transcriptional control of promoters with increased activity in cancer cells. Moreover, the activity of such promoters can be characterized by strong tissue specificity, that is, be specific for certain types of tissues (respectively, and certain types of cancer). This property is especially useful in treating cancer metastases of one nature in tissue of a different origin (for example, breast cancer metastases to bone tissue (Rabbani, SA, and Mazar, AP (2007). Evaluating distant metastases in breast cancer: from biology to outcomes. Cancer Metastasis Rev 26, 663-74)). Extremely important is the “fine tuning” of the gene promoter system, which should ensure the harmlessness of the gene therapeutic drug for normal body tissues. In other words, the “leakage” of the promoter should be excluded at a level that ensures the expression of the killer gene at a level sufficient for the therapeutic effect in healthy cells. For this, in some cases, the used promoter sequences are modified by removing or introducing into their composition regulatory sequences, as a rule, recognition sites of transcription factors. This allows you to modulate the tissue specificity of the expression of the created genetic engineering constructs.

At the same time, none of the above systems can provide strong, strictly tissue-specific expression of the effector gene in germ cells.

Tissue-specific gene expression in germ cells (HA) is due to the presence of a number of unique transcription factors in this type of cells that recognize specific nucleotide motifs in the promoters of regulated genes. The specificity of the action of a larger number of such transcription factors has not yet been established, but for promoters of a significant number of genes, their tissue-specific activation is known precisely in mammalian HA (Wu, H., Chen, Y., Miao, S., Zhang, C., Zong, S. , Koide, SS, and Wang, L. Sperm associated antigen 8 (SPAG8), a novel regulator of activator of CREM in testis during spermatogenesis. FEBS Lett 584, 2807-15). Moreover, the characteristic of regulatory elements in the composition of such promoters, as a rule, has not been carried out, and the exact boundaries of the promoters have not been determined. For example, for the human MAST2 gene, which encodes a special form of serine / threonine kinase associated with microtubules, the pronounced tissue specificity of its expression in germline cells is known, but the regulation of gene expression has not been studied and the promoter sequence has not been functionally characterized. It is also known that promoters of certain repeating elements of the genome are activated in HA, in particular, long terminal repeats of human endogenous retroviruses of the HERV-K family (HML-2) (Buzdin, A. (2007). Human-specific endogenous retroviruses. Scientific World Journal 7, 1848 -68). As in the previous case, the range of transcription factors responsible for the regulation of these elements in HA is unknown.

A number of promoters, mainly active in HA, were patented to obtain various tissue-specific genetic constructs. Thus, the survivin gene promoter can be used for preferential transcription of genetically engineered cassettes in cancer cells of various etiologies (Jyunichi, 2009).

However, such promoters have significant drawbacks: they either demonstrate significant transcriptional activity in other types of cells than HA, and / or cannot provide high transcription of the underlying genes in the introduced constructs.

Promoters for tissue-specific gene expression in HA are highly sought after. For example, low-differentiated germline human tumors (about 1% of all detected cases of cancer) lead to the most aggressive types of malignant germinogenic tumors. The creation of therapeutic constructs for their treatment using new highly effective tissue-specific promoters is a promising area of biotechnological and pharmacogenetic studies.

A prototype of the present invention is a model for using a tissue-specific promoter of the human involucrin gene for expression of various kinds of proteins in epithelial cells (United States Patent Application 20050193431; Tissue specific promoters and transgenic mouse for the screening of pharmaceuticals). This model uses the property of a unique fragment of the human genome, which consists in tissue-specific promoter activity. The invention allows to obtain tissue-specific expression of any introduced genes in the epithelium of the kidneys, ureters, bladder, uterus, esophagus, prostate, spleen and bronchi. However, the prototype invention does not allow tissue-specific expression of the introduced genes in the HA.

The invention solves the problem of creating an artificial strong promoter with high tissue specificity of gene expression in HA.

The problem is solved due to the structure of the promoter, consisting of the overlying sequence of the human gene NDUFV1 with a length of 3574 nucleotides with a deleted proximal sequence of 91 bp in length.

The human gene NDUFV1 is a household gene and encodes a protein involved in the implementation of cellular metabolism in mitochondria (Dieteren, C.E., Willems, PH, Vogel, R.O., Swarts, HG, Fransen, J., Roepman, R. , Crienen, G., Smeitink, JA, Nijtmans, LG, and Koopman, WJ (2008). Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells. J Biol Chem 283, 34753-61 ) Both at the RNA level and at the protein level, this gene is expressed in all human tissues. The transcriptional regulation of this gene is carried out by an overlying (relative to the point of transcription start) sequence, which includes a promoter and a number of distant regulatory elements. The transient transfection experiment of plasmid vectors containing an upstream 3665 nucleotide NDUFV1 gene regulatory sequence shows that it provides expression of the luciferase reporter gene at a level higher than the level of lucifenase expression for the early promoter of SV40 virus, in most of the twelve tested human origin of different cell lines (Figure 1).

The following cell lines are used: EP2102, Tera1 (undifferentiated germinal testicular cancer), Tera-2 (germinal testicular cancer, partially differentiated into neural tube cells), NGP127 (neuroblastoma cells), HepG2 (hepatocarcinoma), A549 (lung carcinoma) and HEK 293 (embryonic kidney cancer). In addition, primary human cell cultures are used: peripheral blood macrophages, umbilical cord mesenchymal stem cells (UC-MSC), placental mesenchymal stem cells (PMSC) and bone marrow mesenchymal stem cells (BM-MSC), as well as transformed germ cells (FBC) )

Removal of 91 pairs of nucleotides located at the 3'-end of this overlying sequence leads to a sharp decrease in the expression of the reporter gene in all cell lines, except for the human germinal testicular cancer lines Tera 1 and EP-2102 (Figure 2). In these lines, which are derivatives of transformed, low-differentiated cells of the germinal pathway, a decrease in the expression level of the reporter gene by ~ 2.9-2.4 times to values higher than those for the early promoter of the SV40 virus is recorded, while in other cell lines the expression level fell by 15 -80 times to hard-to-detect values.

Such a shortened regulatory sequence with a length of 3574 nucleotides ensures the expression of the reporter gene in the Tera 1 cell line at a high level corresponding to the standard CMV promoter and about two times higher than the level of the SV40 promoter, while expression in the remaining lines is detected only at a negligible level (Figure 3 )

A map of potential transcription factor binding sites constructed using AliBaba program and TRANSFAC database (Figure 4) shows that the highest frequency of such sites is in the sequence of the single long terminal repeat of the endogenous HERV-K family (HML) located at the 5'-end of the promoter -2). Further experiments compare the transcriptional activity of the original promoter with a length of 3574 nucleotides and a variant of the promoter carrying a deletion in the complete sequence of the long terminal repeat of HERV-K (HML-2). When the HERV-K (HML-2) element is removed, the promoter activity drops 3-4 times in Tera-1 and EP2102 cell lines, and remains unchanged in the rest of the tested cell lines. Thus, the HERV-K (HML-2) sequence is an important regulatory element of the promoter, which acts as a tissue-specific enhancer.

The promoter is tested on a model of transgenic animals. Three lines of BALB / C mice were created that carry a cassette inserted into the genomic DNA, including the promoter and the gene for red fluorescent protein Ds-Red (Clontech) under its transcriptional control. Subsequently, microsections of tissues homozygous and heterozygous for the presence of an insert of mice are analyzed using a fluorescence microscope. It was found that in both homo- and heterozygous animals, the promoter provided a strong level of expression of the fluorescent protein gene in the testes, comparable to the level of expression of the GAPDH gene (mainly in HA) and, to a lesser extent, in the ovaries, while gene expression in other tissues observed at background level.

The promoter (SEQ ID NO 1) can be used for tissue-specific gene expression in human and mammalian germ cells. Based on the promoter sequence, genetic engineering constructs can be created for research needs, for targeted differentiation of cells, and for gene therapy of various diseases, for example, cancer of germinal tissues.

By further genetic engineering manipulations, the size and composition of the base promoter with a length of 3574 nucleotides can be changed to achieve additional or special goals, such as obtaining a more compact regulatory sequence, increasing or decreasing the level of tissue specificity, introducing additional sites for the binding of transcription factors, introducing restriction sites , the introduction of additional gene sequences, and others.

Based on the promoter sequence, plasmid, viral, or other vector constructs can be created for tissue-specific expression of any gene or various genes in germinal cells, embryonic and stem cells. Moreover, the size of the promoter can be increased, can be reduced to a total value of 400 pairs of nucleotides, and the nucleotide sequence can be changed to a final level of identity of 80% or more with the sequence (SEQ ID NO 1). A sequence can be introduced as a single promoter, or as an additional promoter, or as part of a genetic engineering construct, other regulatory and genetic elements can be added to it.

The invention is illustrated by graphic materials.

Figure 1. Activity of the firefly luciferase reporter gene activity in transfected cell lines.

The activity of the reporter gene is measured using the double luciferase method and normalized to the activity of the control vector pRL-TK (Promega) containing the herpes simplex virus thymidine kinase promoter. The abscissa shows the normalized activity of firefly luciferase. Black bars correspond to transfection with a vector containing the late promoter of the SV40 virus (PV) before the luciferase gene; the light columns correspond to transfection with a vector containing a regulatory sequence of 3665 nucleotide pairs of the human gene ND UFV1 (NUS). Transfection is performed for human cell lines of the following origin: germ cell tumors - undifferentiated (Tera1 line) and highly differentiated (Tera2), neuroglial tumor (NGP127), liver cancer (HepG2), lung cancer (A549), primary macrophage culture, primary culture of placental fibroblasts (FBTs), primary cord blood fibroblast culture (PCF), primary bone marrow cell (BM) culture, fetal kidney cancer (HEK293). For all lines except HEK293, transfection was performed in 6 replicates during two independent experiments, for the HEK293 line, in 4 replicates during one experiment. A generally stronger promoter activity of the NDUFV1 promoter is seen compared to the SV40 virus promoter.

Figure 2. Activity of the firefly luciferase reporter gene activity in transfected cell lines.

The abscissa shows the normalized activity of firefly luciferase. The columns correspond to transfection with a vector containing, in front of the luciferase gene, a modified regulatory sequence of the human gene NDUFV1 (mNUS) with a length of 3574 nucleotides. The presence of significant promoter activity of mNUS, comparable to the activity of the CMV promoter, is observed only in Tera-1 and EP2102 cell lines.

Figure 3. Scheme of the overlying region of the human NDUFV1 gene.

The 3665 nucleotide length NUS sequence contains a highly conserved 91 bp region located immediately above the transcription start point of the NDUFV1 gene. At the 5'-end of the NUS sequence, there is a single terminal repeat of the endogenous retrovirus HERV-K (HML-2).

Figure 4. Scheme of the predicted binding sites of transcription factors within the entire NUS sequence of 3665 bp.

Separately shown is the HERV-K element (HML-2) and a conservative sequence of 91 bp long. The signs above and below the lines indicate the predicted binding sites of transcription factors in the forward and reverse orientations, respectively. Dark ovals indicate the predicted binding sites of the transcription factor SRY / SOX3 corresponding to the consensus sequence ACAAAACA.

Figure 5. Comparison of the activity of the promoter with a length of 3574 nucleotide pairs (mNUS) in a transposition test using the Sleeping Beauty transposon system on Tera-1 and HEK293 cell lines.

(A) General outline of the transposition test. Only cells that receive the genomic insertion of the puromycin resistance cassette can divide and form colonies on an antibiotic medium. In case of random genomic inserts not related to the SB 100 system, the number of colonies is ten times lower than the number of colonies observed with high activity of the SB100X transposase gene. (B) Representative screening of the number of colonies. For each type of experiment, under the representative Petri dishes, the counted numbers of colonies are shown.

Measurement of the proportion of living cells in experiments with the Tera-1 and HEK-293 lines. Experiment ^a Tera-1 HEK-293 PI test ^b Annexin V ^c PI test ^b Annexation V ^c -5FC; -Transf. 9.2 ± 2.3 Nd 7.7 ± 1.5 Nd -5FC; n / a-CDA 9.0 ± 1.7 Nd 7.4 ± 1.7 Nd -5FC; mNUS-CDA 8.8 ± 2.9 Nd 7.8 ± 2.1 Nd -5FC; CMV-CDA 9.9 ± 2.5 Nd 8.2 ± 2.2 Nd + 5FC; - Transfer. 9.4 ± 2.1 10.9 ± 3.3 7.4 ± 1.8 8.7 ± 2.3 + 5FC; n / a-CDA 9.1 ± 2.5 10.7 ± 2.6 7.7 ± 1.6 8.6 ± 2.9 + 5FC; mNUS-CDA 59.5 ± 6.4 59.1 ± 3.9 7.8 ± 1.7 8.4 ± 2.8 + 5FC; CMV-CDA 67.3 ± 7.5 64.9 ± 5.6 73.8 ± 11.3 75.5 ± 8.6 ^a Cell viability measurements were taken 24 hours after the addition of 5-fluorocytosine (5FC). "-5FC" indicates the absence of 5FC in the culture medium, "+ 5FC" indicates experiments with 5FC added to the medium. “Transf.”, “N / p-CDA”, “mNUS-CDA” and “CMV-CDA” mean respectively “no transfection”, “transfection with a construct carrying the cytosine deaminase (CDA) gene, but lacking a promoter” , "transfection with mNUS-CDA construct" and "transfection with SMU-CDA construct". ^b Tests using propidium iodide (PI) were done in four replicates. The values obtained reflect the joint proportion of necrotic and apoptotic cells. ^c Tests using annexin V were performed in triplicate for all experiments with 5FC added to the medium. The values obtained reflect the joint proportion of necrotic and apoptotic cells.

The invention is illustrated by examples.

Example 1

a) cloning the promoter sequence into the pGL3 basic vector (Promega). The promoter sequence is PCR amplified from a human genomic DNA template using primers: NDfor, ACGCGTTGAGTATGCTGCAGGCTTGG; NDrev, AGATCTGCTGCGCCCCTTCAACTTCGCC, is cloned into the pGL3 basic vector in direct orientation upstream of the luciferase gene using restriction sites of the MluI and BglII enzymes. The primary structure of the cloned promoter was verified by direct sequencing and turned out to be 100% identical to the reference sequence of the human genome (genomic contig located in GenBank with access code NT_167190.1, coordinates 12676563-12680136).

b) testing the resulting vector on cell lines.

A series of transient transfections is performed using the Lipofectamine 2000 (Invitrogen) or Unifectin 5000 (Rusbiolink) transfection reagent. Cells of the studied cultures (EP2102, Tera1 (undifferentiated germinal testicular cancer), Tera-2 (germinal testicular cancer, partially differentiated into neural tube cells), NGP127 (neuroblastoma cells), HepG2 (hepatocarcinoma), A549 (lung carcinoma) and HEK 293 embryonic kidney cancer), peripheral blood macrophages, umbilical cord mesenchymal stem cells (UC-MSC), placental mesenchymal stem cells (PMSC) and bone marrow mesenchymal stem cells (BM-MSC), as well as transformed germ cell (FBC) cells 24-hole plats nshet. For screening luciferase activity, use the Dual luciferase system (Promega) kit. For each transfection, use 0.5 μg of the mixture in a 10: 1 ratio of the analytical plasmid (containing the studied promoter above the firefly luciferase gene) and the normalization plasmid. To normalize, use the plasmid pRL-TK (Promega), containing another reporter gene - luciferase Renilla reniformis, under the control of the herpes simplex virus thymidine kinase promoter. The pRL-TK vector provides a constant level of expression of Renilla luciferase in various types of human cell lines. Both luciferase activities are measured only for experiments with transfection efficiency lying in the range of ~ 50-85%. Transfection efficiency is measured by cotransfection with the pTurbo-GFP vector (Evrogen), by measuring the fluorescence of a green fluorescent protein. 24 hours after transfection, the cells are lysed and the firefly and Renilla luciferase activities measured using the Dual-Luciferase Reporter Assay System (Promega) on a GENios Pro luminometer (Tecan). To minimize the error value, the obtained values for firefly luciferase are normalized to Renilla luciferase values. Each experiment is carried out in at least four replicates.

c) Results.

As a result, it was found that of the 12 types of cell cultures, the studied promoter has promoter activity only in HA. In HA, its promoter strength is 2-2.5 times greater than the strength of the SV40 promoter. Tissue-specific expression of luciferase can be useful in co-cultivation of various types of cells for labeling HA, as well as in the production of stably transfected cell lines and development of mechanisms for their directed differentiation for the needs of cell biology or tissue engineering.

Example 2. The use of a promoter for tissue-specific expression of the gene of recombinant cytosine deaminase in HA

a) cloning of the recombinant cytosine deaminase gene under the control of the promoter under study.

The bifunctional recombinant cytosine deaminase (RCD) gene is a gene for the nucleic acid metabolism enzyme that is effectively used in suicidal tumor therapy. In the presence of a protein product of this gene, 5-fluorocytosine, in the medium of a non-toxic specific substrate, the enzyme turns it into a set of highly toxic products for the cell. Due to the ability of toxic metabolites to leave the cell and penetrate into neighboring cells, the effect is not only on cells that received and expressing RCD, but also on neighboring cells, which significantly increases the therapeutic value of the gene. RCD gene, previously in the work (Graepler, F., Lemken, ML, Wybranietz, WA, Schmidt, U., Smimow, I., Gross, CD, Spiegel, M., Schenk, A., Graf, H., Lauer , UA, Vonthein, R., Gregor, M., Armeanu, S., Bitzer, M., and Lauer, UM (2005). Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model. World J Gastroenterol 11, 6910-9) named "SuperCD suicide fusion gene", is cloned according to the experimental protocol published in this work. The RCD gene is placed under the control of the studied promoter in the vector according to paragraph a) of Example 1 using restriction sites of NotI-SalI enzymes. Similarly, the gene is cloned under the control of the CMV cytomegalovirus promoter and into the pGL3 basic vector (Promega), which does not carry any promoter, using the HindIII and NotI restriction sites. The identity of the RCD gene of the expected sequence in all constructs is confirmed by complete direct sequencing of plasmid vectors.

b) testing the resulting vectors with the RCD gene on cell lines.

A series of transient transfections was performed using the transfection reagent Unifectin 2000 (Rusbiolink). Cells of the studied cultures (in this example, Tera1 (undifferentiated germinal testicular cancer) and HEK 293 (embryonic kidney cancer) were plated on 6-well plates. RCD expression was confirmed by Western blot analysis with polyclonal sheep antibodies against the RCD fragment (Abeam) after 24 hours after transfections Functional testing is performed as follows: cells are cotransfected with a 10: 1 mixture from a vector containing RCD and pTurbo-GFP (Evrogen) vector. pTurbo-GFP serves as a normalization vector for assessing transfection efficiency. only experimental results with transfection efficiencies in the range of 65-80% are taken. 12 hours after transfections, medium containing 500 μM 5-fluorocytosine (5FC) is added to the cells. Cell viability was analyzed 24 hours after adding 5FC medium. Each experiment was repeated in four replicates for a test using propidium iodide (PI) and in three replicates for annexin V test (Annexin V test). The results of the PI test are analyzed on a flow cytometer according to a standard protocol (for example, as described in (Kholodenko, R., Kholodenko, I., Sorokin, V., Tolmazova, A., Sazonova, O., and Buzdin, A. ( 2007) .Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism. Cell Res 17, 151-62). Annexation V test and counting dead and apoptotic cells are performed using the Annexin V kit ( Caltag Laboratories) according to the manufacturer's recommendations using a Coulter EPICS XL flow cytometer (Beckman Coulter).

c) Results.

It was shown that the studied promoter at a high level (comparable to that for the CMV promoter) determines the expression of the RCD gene only in HA, while in the control cell line HEK 293 the RCD product is not produced under its control. In HA, its promoter strength is about 85-90% of the CMV promoter strength. Cells expressing RCD are sensitive to 5FC and are sent to apoptosis, unlike most cells not expressing RCD (Table 1). An example shows the possibility of using a promoter for targeted therapy of HA tumors, such as seminoma. The therapeutic construct, which may include RCD or any other gene, is active only in HA (for example, in cells of germinal cancer), but is harmless to other tissues.

Example 3. The use of a promoter for tissue-specific stable transfection of HA

a) Cloning of the Sleeping Beauty transposase gene under the control of the promoter under study.

From the vector based on the pGL3 basic plasmid according to paragraph 1 a) of Example 1 containing the firefly luciferase gene under the control of the promoter under investigation, using the BglII u XbaI restriction endonucleases, the luciferase gene is replaced with the Sleeping Beauty transposon DNA transposase gene, a variant of the translase gene pCMV SBlOOX (Mates, L., Chuah, MK, Belay, E., Jerchow, B., Manoj, N., Acosta-Sanchez, A., Grzela, DP, Schmitt, A., Becker, K., Matrai, J ., Ma, L., Samara-Kuko, E., Gysemans, C., Pryputniewicz, D., Miskey, C., Fletcher, B., Vandendriessche, T., Ivies, Z., and Izsvak, Z. ( 2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41, 753-61).

Transposase highly efficiently embeds DNA fragments of various lengths into the genome, limited by special sequences recognized by it - the so-called inverted terminal repeats (ITR, inverted terminal repeats). A cassette designed for insertion into the genome and limited by ITR is delivered to the cell as part of a plasmid vector. With the simultaneous delivery of the vector and transposase expression in the cell, the cassette is cut out of the vector and inserted into an arbitrary location in the genome of the host cell. Thus, stable transfection of cell cultures, as well as the manufacture of transgenic animals, is highly effective.

b) Obtaining stably transfected HA lines.

Cells of the HA-related lines: Tera-1 and EP2102, as well as the HEK293 lines (control cells), are plated on 6-well plastic culture plates and grown to reach a 70-100% level of confluency. Then the cells are transfected with a set of plasmids using the transfection reagents Unifectin 2000 (Rusbiolink) and FuGENE (Roche). The following vectors are used: pTIB / puro, including the limited ITR antibiotic resistance gene puromycin under the control of the SV40 promoter; pCMV-SB100X containing the SB100X gene under the control of the CMV promoter; pNo-SB100X not containing a promoter in front of the transposase gene; pMNUS-SB100X containing the SB100X gene under the control of the promoter under study; pTurboGFP (Evrogen), comprising the GFP gene under the control of the CMV promoter. Transfection efficiency is evaluated by GFP fluorescence. The following combinations of vectors are used: a) only pT2B / puro, b) pT2B / puro + no-SB100X, c) pT2B / puro + CMV-SB100X, d) pT2B / puro + mNUS-SB100X. After transfection, cells are incubated at 37 ° C, 5% CO2, for 48 hours. Cells are washed from the medium and transplanted onto 10 cm Petri dishes for puromycin selection (~ 7 days). 10 ml of puromycin medium is added to each cup. At the end of the selection period, the surviving cells form colonies with a diameter of 2-3 mm each. Colonies are stained after fixation in 10% formaldehyde in PBS and incubation for 20 minutes at room temperature under traction. Cells were washed twice with PBS and 5 ml of staining solution (0.5% methylene blue in PBS) was added to each dish, then incubated for 30 minutes. The dye is removed, the plates are washed and dried. The efficiency of the formation of stably transfected colonies is considered as the ratio of the number of colonies formed in the presence of transposase to the number of colonies formed without transposase due to random insertion of a vector with a puromycin resistance cassette into the genome.

c) Results.

Judging by the efficiency of the formation of stably transfected colonies (Figure 5), it was found that the promoter under study directs the expression of the SB100X gene only in HA, but not in control cells. The strength of the promoter is comparable to the CMV promoter. Thus, this example clearly demonstrates that the promoter under study can be used for effective selective stable transfection of HAs by any genetic cassettes.

Example 4. Obtaining transgenic animals expressing the fluorescent protein gene in HA and related tissues and organs

According to the methodology described in detail in the article (Zaitseva, I., Zaitsev, S., Alenina, N .. Bader, M., and Krivokharchenko, A. (2007). Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro. Mol Reprod Dev 74, 1255-61), transgenic lines of BALB / C mice were obtained. In this case, the plasmid construct according to point a) from Example 1 is introduced into the oocytes in mice, containing, under the control of the promoter under study, the Ds-red fluorescent protein gene (Clontech, USA) instead of the firefly luciferase gene. The mice in whose genome the insertion occurred were determined from the first litter during PCR analysis of genomic DNA isolated from mouse tails by the appearance of new genetic markers. A total of three new transgenic mouse lines were obtained. Then, for animals homo- and heterozygous for the genomic insert of the above construction, microscopic examination of histological sections is performed. In all cases, for males in the testes, fluorescence characteristic of the Ds-red protein was observed, while in other organs comparable levels of fluorescence were not observed. In females, fluorescence in the ovary region was weaker than in males in the testes, against the background of a lack of fluorescence in other organs.

The example proves the applicability of this promoter for the creation of transgenic animals with characteristic profiles of tissue-specific gene expression in HA. In addition, the example shows the possibility of using a promoter for gene therapy, in terms of the absence of side effects for other than target tissues and organs.

Claims (1)

A promoter for tissue-specific gene expression in mammalian germ tissues, consisting of a 3574-long nucleotide sequence located above the transcription start point of the human NDUFV1 gene with a 91 bp deleted sequence located immediately above the transcription start point of this gene.

Images