In Vitro Evidence of Selective Pro-Apoptotic Action of the Pure Cannabidiol and Cannabidiol-Rich Extract.

Plant cannabinoids, secondary metabolites of species belonging to the Cannabis genus, can mimic the endocannabinoids' action and exert biological effects. Considering the contribution of the endocannabinoid system in cell cycle and apoptotic regulation, there is an interest in exploring the potential anti-cancer activities of natural and synthetic cannabinoids. Cannabidiol (CBD), an abundant plant cannabinoid, reveals a low affinity to cannabinoid receptors and, contrary to various cannabinoids, lacks psychoactive action. Here, we present the in vitro assessment of the pro-apoptototic potential of CBD-rich extracts of Cannabis sativa L. (eCBD) compared to purified CBD (pCBD). As demonstrated, both eCBD and pCBD decreased the viability of breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC-3 in a concentration-dependent fashion. Endoplasmic reticulum stress-related apoptosis and morphological changes were induced only in low-serum conditions. Moreover, the effects of eCDB and pCDB were also assessed in non-malignant cell lines (MCF-10A and PNT2) with no alterations of viability noted, ultimately suggesting a selective action of CBD in tumor cells. The results suggest the possible involvement of reactive oxygen species in the response mechanism to eCBD and pCBD, but no clear pattern was observed. We also demonstrated significant changes in gene expression involved in apoptosis and cell cycle control upon extract treatment. Altogether, our study shows the potential of eCBD and pCBD as novel pro-apoptototic agents that can be considered promising in future preclinical and clinical testing.

Effects of 2.4 GHz radiofrequency electromagnetic field (RF-EMF) on glioblastoma cells (U -118 MG).

INTRODUCTION AND OBJECTIVE: Mobile phones and Wi-Fi are the most commonly used forms of telecommunications. Initiated with the first generation, the mobile telephony is currently in its fifth generation without being screened extensively for any biological effects that it may have on humans or on animals. Some studies indicate that high frequency electromagnetic radiation emitted by mobile phone and Wi-Fi connection can have a negative effect upon human health, and can cause cancer, including brain tumour. OBJECTIVE: The aim of the study was to investigate the influence of 2.4 GHz radiofrequency electromagnetic field (RF-EMF) on the proliferation and morphology of normal (human embryonic kidney cell line Hek-293) and cancer cells (glioblastoma cell line U-118 MG). MATERIAL AND METHODS: The cell cultures were incubated in RF-EMF at the frequency of 2.4 GHz, with or without dielectric screen, for 24, 48 and 72h. In order to analyse the influence of the electromagnetic field on cell lines, Cytotoxicity test Cell Counting Kit-8 was performed. To protect cells against emission of the electromagnetic field, a dielectric screen was used. RESULTS: It was found that 2.4 GHz RF electromagnetic field exposure caused a significant decrease in viability of U-118 MG and Hek-293 cells. The impact of the electromagnetic field was strongest in the case of cancer cells, and the decrease in their survival was much greater compared to the healthy (normal) cells of the Hek-293 line. CONCLUSIONS: Results of the study indicate that using a radio frequency electromagnetic field (2.4 GHz) has a clearly negative effect on the metabolic activity of glioblastoma cells. RF-EMF has much less impact on reducing the viability of normal cells (Hek -293) than cancer cells.

Evolution of CRISPR/Cas Systems for Precise Genome Editing.

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.

No wonder, it is a hybrid. Natural hybridization between Jacobaea vulgaris and J. erucifolia revealed by molecular marker systems and its potential ecological impact.

Progressive changes in the environment are related to modifications of the habitat. Introducing exotic species, and interbreeding between species can lead to processes that in the case of rare species or small populations threatens their integrity. Given the declining trends of many populations due to increased hybridization, early recognition of hybrids becomes important in conservation management. Natural hybridization is prevalent in Jacobaea. There are many naturally occurring interspecific hybrids in this genus, including those between Jacobaea vulgaris and its relatives. Although Jacobaea erucifolia and J. vulgaris often co-occur and are considered closely related, apart from the few reports of German botanists on the existence of such hybrids, there is no information on research confirming hybridization between them. Morphologically intermediate individuals, found in the sympatric distributions of J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems (nuclear and chloroplast DNA markers) were employed to test this hypothesis and characterize putative hybrids. Nuclear and chloroplast DNA sequencing results and taxon-specific amplified fragment length polymorphism (AFLP) fragment distribution analysis confirmed the hybrid nature of all 25 putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to J. erucifolia, suggesting frequent backcrossing. Moreover, they showed that several individuals previously described as pure were probably also of hybrid origin, backcrosses to J. erucifolia and J. vulgaris. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared to be bidirectional but asymmetrical with J. vulgaris as the usual maternal parent.

Influence of Three Laser Wavelengths with Different Power Densities on the Mitochondrial Activity of Human Gingival Fibroblasts in Cell Culture.

Phototherapy plays a key role in wound healing and tissue regeneration. The use of lasers has the potential to become an effective and minimally invasive treatment in periodontal and peri-implant disease. The aim of this study was to evaluate the influence of three laser wavelengths with the combination of parameters such as power density and energy density on human gingival fibroblasts (hGFs) in vitro culture. Isolated cells were seeded in 96-well plates with culture medium (DMEM, Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS). After 24 h cells were irradiated (1064, 980 and 635 nm, various energy density value). After 24, 48 and 72 h, cells were evaluated for viability. Data were analyzed by ANOVA followed by Tukey's HSD test. We found the best outcomes for hGFs irradiated with laser 1064 nm for all combinations of power output (50/400/1000 mW) and energy dose (3/25/64 J/cm2) after 48 h and 72 h compared with control group. Cell viability increase ranged from 0.6× (3 J/cm2, 50 mW) to 1.3× (64 J/cm2, 1000 mW). Our findings indicate that the appropriate use of low-level laser irradiation (LLLI) can increase the proliferation rate of cultured cells. The use of LLLI can be extremely useful in tissue engineering and regenerative medicine.

Frequency and Nuisance Level of Adverse Events in Individuals Receiving Homologous and Heterologous COVID-19 Booster Vaccine.

This study aimed to compare the occurrence and nuisance of adverse events following administration of each COVID-19 vaccine dose between two groups: individuals given three doses of mRNA vaccine (homologous group, 3 × mRNA, n = 252) and those given two doses of adenoviral vector vaccine further boosted with mRNA vaccine (heterologous group, 2 × AZ + 1 × mRNA, n = 205). Although the studied groups differed significantly in the frequency and number of side effects after the first and second vaccine dose, no relevant differences were seen following the booster administration. Arm pain and fatigue were the most common effects, regardless of the vaccination group and vaccine dose. In the homologous group, female sex, lower BMI, and no history of regular influenza vaccination were associated with a higher frequency of side effects of a booster dose. In the heterologous group, the history of COVID-19 was associated with an increased number of side effects seen after a booster. In both groups, the number of side effects related to the first and second dose correlated with the number observed after administration of a booster dose. Individuals receiving a homologous booster reported a higher nuisance of side effects than the heterologous group. It was similar to the level reported after the second dose in both groups. The use of pharmaceuticals to counteract the side effects was more frequent after a first dose in the 2 × AZ + 1 × mRNA group, but higher after second dose in individuals receiving the 3 × mRNA vaccination scheme. The frequency of pharmaceutical use after a booster dose was similar in both groups (approx. 60%). Paracetamol was most frequently chosen, regardless of the group and vaccine dose. In addition, the vast majority of participants (93%) declared to accept future doses of the COVID-19 vaccine if their administration would be recommended. This study provides an overview of the response to homologous and heterologous mRNA vaccine booster dose that may be valuable in shaping accurate and honest communication with vaccinated individuals, especially in those regions which are yet to pursue booster strategies.

The Perception and Attitudes toward COVID-19 Vaccines: A Cross-Sectional Study in Poland.

Vaccine hesitancy is a major threat to the success of COVID-19 vaccination programs. The present cross-sectional online survey of adult Poles (n = 1020) expressing a willingness to receive the COVID-19 vaccine was conducted between February and March 2021 and aimed to assess (i) the general trust in different types of vaccines, (ii) the level of acceptance of the COVID-19 vaccines already in use in Poland (BNT162b2 by BioNTech/Pfizer, mRNA-1273 by Moderna and AZD1222 by Oxford/AstraZeneca) as well as eight vaccines approved outside European Union (EU) or in advanced stages of clinical trials, (iii) level of fear of vaccination against COVID-19, and (iv) main sources of information on COVID-19 vaccination. Among all major vaccine technology, the highest level of trust was observed for the mRNA platform, with a considerable number of surveyed (>20%) not aware of the existence of vaccines produced using the traditional approach (inactivated and live attenuated vaccines). The age of participants was the main factor differentiating the level of trust in a particular vaccine type. Both BNT162b and mRNA-1273 received a high level of acceptance, contrary to AZD1222. From eight vaccines unauthorized in the EU at the moment of study, the CVnCoV (mRNA; CureVac) was met with the highest level of trust, followed by Ad26.COV2.S (vector; Janssen/Johnson&Johnson) and NVX-CoV2373 (protein; Novavax). Sputnik V (vector; Gamaleya Research Institute) was decidedly the least trusted vaccine. The median level of fear (measured by the 10-point Likert-type scale) in the studied group was 4.0, mostly related to the risk of serious allergic reactions, other severe adverse events and unknown long-term effects of vaccination. Female, individuals with a lower level of education and those not seeking any information on the COVID-19 vaccines revealed a higher fear of vaccination. Experts' materials were the major source of information on COVID-19 vaccines in the studied group. The study shows the level of trust in COVID-19 vaccines can vary much across the producers while the mRNA vaccines are received with a high level of acceptance. It also emphasizes the need for effective and continuous science communication when fighting the pandemic as it may be an ideal time to increase the general awareness of vaccines.

CRISPR/Cas Technology in Pig-to-Human Xenotransplantation Research.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats linked to Cas nuclease) technology has revolutionized many aspects of genetic engineering research. Thanks to it, it became possible to study the functions and mechanisms of biology with greater precision, as well as to obtain genetically modified organisms, both prokaryotic and eukaryotic. The changes introduced by the CRISPR/Cas system are based on the repair paths of the single or double strand DNA breaks that cause insertions, deletions, or precise integrations of donor DNA. These changes are crucial for many fields of science, one of which is the use of animals (pigs) as a reservoir of tissues and organs for xenotransplantation into humans. Non-genetically modified animals cannot be used to save human life and health due to acute immunological reactions resulting from the phylogenetic distance of these two species. This review is intended to collect and summarize the advantages as well as achievements of the CRISPR/Cas system in pig-to-human xenotransplantation research. In addition, it demonstrates barriers and limitations that require careful evaluation before attempting to experiment with this technology.

Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation.

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (Sus scrofa domestica) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1, CMAH, ß4GalNT2, vWF, ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.

Application of Genetically Engineered Pigs in Biomedical Research.

Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. Animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. Another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. The further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. Numerous studies have shown that the pig (Sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. Short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species This review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models.

Cannabinoids in Medicine: Cancer, Immunity, and Microbial Diseases.

Recently, there has been a growing interest in the medical applications of Cannabis plants. They owe their unique properties to a group of secondary metabolites known as phytocannabinoids, which are specific for this genus. Phytocannabinoids, and cannabinoids generally, can interact with cannabinoid receptors being part of the endocannabinoid system present in animals. Over the years a growing body of scientific evidence has been gathered, suggesting that these compounds have therapeutic potential. In this article, we review the classification of cannabinoids, the molecular mechanisms of their interaction with animal cells as well as their potential application in the treatment of human diseases. Specifically, we focus on the research concerning the anticancer potential of cannabinoids in preclinical studies, their possible use in cancer treatment and palliative medicine, as well as their influence on the immune system. We also discuss their potential as therapeutic agents in infectious, autoimmune, and gastrointestinal inflammatory diseases. We postulate that the currently ongoing and future clinical trials should be accompanied by research focused on the cellular and molecular response to cannabinoids and Cannabis extracts, which will ultimately allow us to fully understand the mechanism, potency, and safety profile of cannabinoids as single agents and as complementary drugs.

Correction to: The production of UL16-binding protein 1 targeted pigs using CRISPR technology.

[This corrects the article DOI: 10.1007/s13205-018-1107-4.].

The current state and future perspectives of cannabinoids in cancer biology.

To date, cannabinoids have been allowed in the palliative medicine due to their analgesic and antiemetic effects, but increasing number of preclinical studies indicates their anticancer properties. Cannabinoids exhibit their action by a modulation of the signaling pathways crucial in the control of cell proliferation and survival. Many in vitro and in vivo experiments have shown that cannabinoids inhibit proliferation of cancer cells, stimulate autophagy and apoptosis, and have also a potential to inhibit angiogenesis and metastasis. In this review, we present an actual state of knowledge regarding molecular mechanisms of cannabinoids' anticancer action, but we discuss also aspects that are still not fully understood such as the role of the endocannabinoid system in a carcinogenesis, the impact of cannabinoids on the immune system in the context of cancer development, or the cases of a stimulation of cancer cells' proliferation by cannabinoids. The review includes also a summary of currently ongoing clinical trials evaluating the safety and efficacy of cannabinoids as anticancer agents.

Genetically Modified Pigs as Organ Donors for Xenotransplantation.

The growing shortage of available organs is a major problem in transplantology. Thus, new and alternative sources of organs need to be found. One promising solution could be xenotransplantation, i.e., the use of animal cells, tissues and organs. The domestic pig is the optimum donor for such transplants. However, xenogeneic transplantation from pigs to humans involves high immune incompatibility and a complex rejection process. The rapid development of genetic engineering techniques enables genome modifications in pigs that reduce the cross-species immune barrier.

CRISPR/Cas9 Immune System as a Tool for Genome Engineering.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) adaptive immune systems constitute a bacterial defence against invading nucleic acids derived from bacteriophages or plasmids. This prokaryotic system was adapted in molecular biology and became one of the most powerful and versatile platforms for genome engineering. CRISPR/Cas9 is a simple and rapid tool which enables the efficient modification of endogenous genes in various species and cell types. Moreover, a modified version of the CRISPR/Cas9 system with transcriptional repressors or activators allows robust transcription repression or activation of target genes. The simplicity of CRISPR/Cas9 has resulted in the widespread use of this technology in many fields, including basic research, biotechnology and biomedicine.

Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.

1,3-Propanediol production by Escherichia coli using genes from Citrobacter freundii atcc 8090.

Compared with chemical synthesis, fermentation has the advantage of mass production at low cost, and has been used in the production of various industrial chemicals. As a valuable organic compound, 1,3-propanediol (1,3-PDO) has numerous applications in the production of polymers, lubricants, cosmetics and medicines. Here, conversion of glycerol (a renewable substrate and waste from biodiesel production) to 1,3-PDO by E. coli bacterial strain carrying altered glycerol metabolic pathway was investigated. Two gene constructs containing the 1,3-PDO operon from Citrobacter freundii (pCF1 and pCF2) were used to transform the bacteria. The pCF1 gene expression construct contained dhaBCE genes encoding the three subunits of glycerol dehydratase, dhaF encoding the large subunit of the glycerol dehydratase reactivation factor and dhaG encoding the small subunit of the glycerol dehydratase reactivating factor. The pCF2 gene expression construct contained the dhaT gene encoding the 1,3-propanediol dehydrogenase. Expression of the genes cloned in the above constructs was under regulation of the T7lac promoter. RT-PCR, SDS-PAGE analyses and functional tests confirmed that 1,3-PDO synthesis pathway genes were expressed at the RNA and protein levels, and worked flawlessly in the heterologous host. In a batch flask culture, in a short time applied just to identify the 1,3-PDO in a preliminary study, the recombinant E. coli bacteria produced 1.53 g/L of 1,3-PDO, using 21.2 g/L of glycerol in 72 h. In the Sartorius Biostat B Plus reactor, they produced 11.7 g/L of 1,3-PDO using 24.2 g/L of glycerol, attaining an efficiency of 0.58 [mol1,3-PDO/molglycerol].

Screening pigs for xenotransplantation: expression of porcine endogenous retroviruses in transgenic pig skin.

Pigs seem to be the answer to worldwide organ donor shortage. Porcine skin may also be applied as a dressing for severe burns. Genetic modifications of donor animals enable reduction of immune response, which prolongs xenograft survival as temporary biological dressing and allows achieving resistance against xenograft rejection. The risk posed by porcine endogenous retroviruses (PERVs) cannot be eliminated by breeding animals under specific-pathogen-free conditions and so all recipients of porcine graft will be exposed to PERVs. Therefore our study has been focused on the assessment of PERV DNA and mRNA level in skin samples of transgenic pigs generated for xenotransplantation. Porcine skin fragments were obtained from 3- to 6-month-old non-transgenic and transgenic Polish Landrace pigs. Transgenic pigs were produced by pronuclear DNA microinjection and were developed to express the human α-galactosidase and the human α-1,2-fucosyltransferase gene. The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR. Comparative analysis of all PERV subtypes revealed that PERV-A is the main subtype of PERVs in analyzed skin samples. There was no significantly different copy number of PERV-A, PERV-B and PERV-C between non-transgenic pigs, pigs with the human α-galactosidase and pigs expressing the human α-1,2-fucosyltransferase gene, except of PERV-C DNA. It brings the conclusion, that transgenesis process exerts no influence on PERVs transinfection. That is another step forward in the development of pig skin xenografts as burn wounds dressing.

1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol1,3-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming there is an increasing industrial need to exchange the chemical way of chemicals synthesis for biotechnological - more ecological manner. The 1,3-PDO production from glycerol is an desirable alternative to the traditional production from non-renewable resources. This work is a part of project, which opens a way to development of innovative "green chemistry" and new perspectives to chemical industry.

Immune modulation in xenotransplantation.

The use of animals as donors of tissues and organs for xenotransplantations may help in meeting the increasing demand for organs for human transplantations. Clinical studies indicate that the domestic pig best satisfies the criteria of organ suitability for xenotransplantation. However, the considerable phylogenetic distance between humans and the pig causes tremendous immunological problems after transplantation, thus genetic modifications need to be introduced to the porcine genome, with the aim of reducing xenotransplant immunogenicity. Advances in genetic engineering have facilitated the incorporation of human genes regulating the complement into the porcine genome, knockout of the gene encoding the formation of the Gal antigen (α1,3-galactosyltransferase) or modification of surface proteins in donor cells. The next step is two-fold. Firstly, to inhibit processes of cell-mediated xenograft rejection, involving natural killer cells and macrophages. Secondly, to inhibit rejection caused by the incompatibility of proteins participating in the regulation of the coagulation system, which leads to a disruption of the equilibrium in pro- and anti-coagulant activity. Only a simultaneous incorporation of several gene constructs will make it possible to produce multitransgenic animals whose organs, when transplanted to human recipients, would be resistant to hyperacute and delayed xenograft rejection.