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Recombinant adenoviruses are widely used for delivery and expression of transgenes for therapeutic purposes and in fundamental research. AdEasy™ adenoviral vector system allows for rapid and efficient generation of replication-deficient adenoviruses. One important crucial step in producing recombinant adenoviruses is the generation of a recombinant adenoviral plasmid in E. coli BJ5183 cells via homologous recombination between the transgene encoding shuttle vector and the pAdEasy-1 plasmid, which carries a significant part of human adenovirus serotype 5 (Ad5) genome. In many published protocols, the necessity of electroporation for the transformation of the shuttle vector and pAdEasy-1 into E. coli cells is emphasized, thus making necessary the availability of the appropriate instruments and consumables. Here, we have proposed an optimized protocol for the generation of recombinant adenoviruses with the use of chemically competent E. coli cells only, without the use of electroporation. We have demonstrated the efficient transformation of linearized nonpurified shuttle vector into chemically competent E. coli BJ5183-pAdEasy-1 cells allowing identification of clones with correct recombinant adenoviral plasmids. Using the optimized protocol, we have generated four functional replication-deficient adenoviruses expressing target proteins in HEK293 cells. The protocol that has been suggested provides a significantly simplified and cheaper method for the generation of recombinant adenoviruses due to lack of requirement to use instruments and consumables for electroporation.

A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter. XRE elements can be used for nonspecific transcription enhancement but are unsuitable for the creation of tumor-specific promoters with enhanced activity.

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase; uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics.

The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk. The current state and prospects for the development of this area of biotechnology are discussed in relation to the emergence of novel genome editing technologies. Experimental and practical techniques are briefly discussed.

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene pre-mRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter. XRE elements can be used for nonspecific transcription enhancement but are unsuitable for the creation of tumor-specific promoters with enhanced activity.

describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human TERT gene promoter (hTERT) and the antioxidant response element (ARE) from the human GCLM gene promoter. The hybrid promoter retains the tumor specificity of the basal hTERT promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the in vitro enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter. The developed hybrid promoter can be considered a better alternative to the hTERT promoter in cancer gene therapy schemes.

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.

Cattle of polled phenotype is convenient for breeders, as it decreases the risk of animals being hurt and ensures safety of workers. We developed the system for editing cattle genome using CRISPR/Cas9 which will allow production of animals with polled phenotype genetically based on any cattle breed without changing its main phenotypic traits.

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

Adsorption calorimetric data on the reactions of liquid phase hydrogenation are used within the surface model with discrete inhomogeneity in the approximation of an ideal adsorption layer to calculate thermodynamic characteristics of equilibria of individual adsorbed hydrogen forms on a partially deactivated nickel catalyst in aqueous solutions of sodium hydroxide of different concentration. The effect of solution composition on the limiting adsorption values and adsorption coefficients of surface hydrogen-metal complexes on a skeletal nickel catalyst is shown.

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene pre-mRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

Thermodynamic characteristics of the adsorption equilibria of individual forms of adsorbed hydrogen are calculated using data from adsorption-calorimetric studies of liquid-phase hydrogenation in a model of a surface with discrete nonuniformity in the ideal adsorption layer approximation under conditions of liquid-phase hydrogenation. It is shown that partial deactivation raises the fraction of the molecular α form via the atomic β form of adsorbed hydrogen and increases the adsorbate-adsorbent binding energy.

Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.

Inactivation of tumor suppressors and activation of protooncogenes are critical events in malignant cell transformation and tumor progression. Pdcd4 encodes a protein with tumor suppressor functions, which accounts for an increased interest to Pdcd4 as a potential diagnostic and prognostic marker, as well as a target for antineoplastic therapy. This review summarizes well-known properties and functions of Pdcd4 tumor suppressor and mechanisms of its regulation in tumor cells. It is also focused to the role of Pdcd4 in cellular transformation and tumor progression, as well as on its potential practical application in oncology.

The effects of the nature and composition of solvents on the thermodynamic characteristics of the adsorption states of hydrogen on nickel were studied. The adsorption can be described in terms of the thermodynamic model of a surface with a discrete nonhomogeneity for three individual forms of adsorbed hydrogen. The thermodynamic characteristics of the individual forms of hydrogen adsorbed on porous nickel from aqueous solutions of sodium hydroxide, dimethylformamide, methanol, and ethanol were determined.

An analysis was made of the change in the expression of the extracellular inhibitor of the Wnt-signal pathway WIF1 in non-small cell lung carcinomas. The results of the analysis revealed a frequent (67% of the cases) suppression of the WIF1 transcript in non-small cell lung carcinomas. On the basis of the data obtained and the results of previously published investigations, it was suggested that inhibition of WIF1 expression is typical of squamous cell carcinoma of the lungs and only to a lesser degree of adenocarcinoma. An analysis of the content of the WIF1 protein in tumor lysates with a suppressed level of the WIF1 transcript also revealed an accompanying decrease in the level of the WIF1 protein in the tumors. The results of the investigation confirm the frequent suppression both of the transcript and of the WIF protein in lung tumors, which may cause a disregulation of the Wnt-signal pathway and may contribute to the development of the tumor.

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.

p21-activated kinases (Paks) are a highly conserved family of enzymes that bind to and are activated by small GTPases of the Cdc42 and Rac families. With the notable exception of plants, nearly all eukaryotes encode one or more Pak genes, indicating an ancient origin and important function for this family of enzymes. Genetic approaches in many different experimental systems, ranging from yeast to mice, are beginning to decipher the different functions of Paks. Although some of these functions are unique to a given organism, certain common themes have emerged, such as the activation of mitogen-activated protein kinase (MAPK) cascades and the regulation of cytoskeletal structure through effects on the actin and tubulin cytoskeletons.

Programmed cell death 4 (Pdcd4) tumor suppressor is frequently lost in tumors of various origins including lung cancer, and its loss contributes to tumor progression. However molecular mechanisms underlying Pdcd4 suppression in lung cancer cells remain largely unexplored. Here we investigated molecular mechanisms of Pdcd4 suppression in lung cancer cells. Besides enhanced mTOR-dependent proteasomal degradation of Pdcd4 protein, we found that Pdcd4 transcription is negatively regulated by mTOR signaling, and localized cis-acting element in Pdcd4 promoter responsible for this effect. In conclusion, we described a novel molecular mechanism of Pdcd4 suppression in cancer cells consisting from mTOR signaling-dependent transcriptional repression of Pdcd4.

Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

Rho family GTPases act as molecular switches to regulate numerous cellular processes, including malignant transformation. Commonly, overexpression of Rho GTPases contributes to tumorigenesis. Elevated expression of several Rho GTPases has been reported in lung cancer and is associated with poor prognosis. The RHOV gene encodes the atypical Rho family GTPase Chp/RhoV, which is capable of transforming fibroblasts, although other functions of Chp remain largely elusive. RHOV is expressed in normal lung tissue in rats, but not in humans. RHOV expression was found in several human cancer cell lines, including non–small-cell lung cancer (NSCLC) cell line A549, but expression of RHOV in NSCLC tumors has never been investigated. Here we studied the expression of the RHOV gene in lung cancer cell lines and in 29 matched pairs of NSCLC tumors and adjacent nontumorous tissues. We found that RHOV is expressed in lung cancer cell lines and is upregulated in the majority of studied lung tumors. Analysis of the Oncomine database revealed correlation between elevated RHOV level and poor patient survival. We propose that the RHOV gene could be validated as a diagnostic or prognostic marker for NSCLC, and that observed overexpression of RHOV might contribute to tumorigenesis.

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.

Efficiency and specificity are two key attributes of anti-cancer drugs including genetic therapeutic agents. We suggest a way to improve specificity of gene therapy drugs based on the ability of 3′-untranslated regions (UTR) of some mRNAs selectively stabilize transcripts only during cell division. The mRNAs of genes encoding DNA methyltransferase I (DNMT1) and topoisomerase IIα (TOP2A) are among such transcripts. When inserted into genetic constructs designed to produce therapeutic protein in tumor cells, such 3′-UTR would lead to diminished effect of therapeutic protein on normal cells, which are characterized by low or absent proliferative activity. However, when included in gene expression cassette, these 3′-UTR might result in decreased transgene expression, thus, overweighting the advantage of increased specificity of expression. We showed that DNMT1 and to the lesser extent TOP2A 3′-UTR do not alter significantly therapeutic transgene expression level in tumor cells, thus, confirming the functionality of the proposed approach.

Inactivation of tumor suppressors and activation of protooncogenes are critical events in malignant cell transformation and tumor progression. Pdcd4 encodes a protein with tumor suppressor functions, which accounts for an increased interest to Pdcd4 as a potential diagnostic and prognostic marker, as well as a target for antineoplastic therapy. This review summarizes well-known properties and functions of Pdcd4 tumor suppressor and mechanisms of its regulation in tumor cells. It is also focused to the role of Pdcd4 in cellular transformation and tumor progression, as well as on its potential practical application in oncology.

Changes in WIFI expression, an extracellular inhibitor of Wnt pathway, in non-small cell lung carcinomas were analyzed. Frequent (67% cases) suppression of WIFI transcript in non-small cell lung carcinomas were found. Our results, together with previously published data, suggest that inhibition of WIFI expression often occurs in squamous cell carcinomas and is less typical of adenocarcinomas. It was also found that a decrease in the WIFI transcript in tumors is parallel to concomitant suppression of the WIFI protein level. Our results provide further evidence that the WIFI suppression is a frequent event in the lung carcinogenesis, which might lead to disregulation of Wnt signaling pathway and contribute to tumor progression.

The author examined the intestinal wall in 5 cadavers of patients deceased from cirrhosis and in 20 animals (cats and dogs) after the ligature of their portal veins. Tissues were treated by Bielschowsky-Gros method with an additional staining of the blood vessels by acid fuchsin. Intimate neurovascular relations were revealed in the liver both in human cirrhosis and in experimental animals (dogs) after the ligature of the portal vein. These relations were represented by the presence of terminations of dendritis of the ganglionic cells on capillaries. Large numbers of synaptic endings of thin fibers (of various length) were also found on the blood vessels.