Genetic modification is a modern approach which allows us to produce organisms with required characteristics, e.g. milk composition [1,2], muscle volume [3,4] or resistance to infectious diseases [5,6]. Today, various species including rodents [7,8], primates [9,10] and cattle [6,11,12] have been genetically modified. The simplest way to create genetically engineered animals with specific properties is to alter the genome of their embryos.

Different techniques are used to achieve this goal: transplantation of genetically modified ESC into the blastocyst cavity followed by formation of mosaic embryo [13,14], injection of RNA or DNA into the zygote or oocyte [[15], [16], [17], [18], [19], [20]], or electroporation of zygotes resulting in the delivery of gene-editing reagents into developing embryos [[21], [22], [23]]. Currently, the efficacy of these methods is widely debated. Moreover, the major drawback of these techniques is that they require expensive equipment and highly qualified operators.

Genetic modification of cells by transduction (including embryos) is a simple method: only basic laboratory equipment (CO2-incubators, microscopes) is needed; it is not necessary to perform microinjections; viruses used to perform transduction can easily be transported almost anywhere in the world. Therefore, production of transgenic embryos can be carried out near farms (where laboratories are often equipped only by basic facilities). Thus, this approach will allow for the execution of all necessary procedures (embryo retrieval, gene modification and embryo transfer to the animal) in one place, reducing the risk of embryo damage during transportation.

Different types of viruses can be utilized to deliver genetic material into the zygote. Retroviruses [24], particularly lentiviruses [25], adenoviruses [26] and adeno-associated viruses [27] are used for these procedures.

Adeno-associated viruses (AAV) are widely used in the field of genetically modified cell and organism production. It has been shown that AAV serve as an effective tool to produce transgenic pig mesenchymal stem cells [28], spermatozoa and embryos [29], adult murine neurons [30], murine [26,27] and bovine embryos [27].

Adeno-associated viruses have significantly smaller virions compared to those of retroviruses or adenoviruses. Their small (approximately 20 nm) size of AAV [31,32], makes penetration of the virion into the embryo easier.

AAV are traditionally considered as a safe viral vector platform due to the fact that the helper virus is required to produce an infection in natural conditions. The adeno-associated virus can infect the cell, but for its reproduction cycle, a helper virus such as Adenovirus or Herpes Simplex virus is required. According to the NIH (National Institutes of Health), all serotypes of recombinant AAV vectors created in the absence of helper viruses are considered as Risk Group 1 agents (agents that are not associated with disease in healthy adult humans) if the transgene does not encode for a toxic or potentially tumorigenic gene. Therefore, many recombinant AAV vectors can be used without additional biosafety concerns [33].

In the current study, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos.

In mammals, CD209 (also known as DC-SIGN; Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) is one of the key proteins involved in multiple immunological events. CD209 is a C-type lectin receptor which is present on the surface of macrophages and dendritic cells [34]. Being a part of the first line of the innate immunity, CD209 recognizes a broad range of pathogens in a rather nonspecific manner: viruses, bacteria, fungi, protists. During coevolution in the host/pathogen systems, some pathogens converted CD209 to their side and used it to spread further (e.g. CD209+ DC are employed to carry HIV virions from the contact site on the mucous membrane to the bloodstream and lymph nodes) and to thus infect different types of cells. The impact of CD209 on the different stages of the infection process was shown for HIV [35], hepatitis C virus [36], Ebola virus, Dengue virus, Coronaviruses, Mycobacterium tuberculosis [37,38], Leishmania [39] and many other infectious agents (reviewed in Ref. [40]).

One possible mechanism of CD209-dependent pathogen protection was elucidated for HIV and hepatitis C viruses. It was shown that, even if normally CD209-bound pathogens are internalized into proteasomes for future antigen presentation, under certain circumstances (such as additional receptors) after binding CD209 HCV and HIV, particles can be internalized into specialized, non-lysosomal, low-pH endosomes [36,41,42]. Recently, it has been demonstrated that CD209 knockout mice are less susceptible to the Toxoplasma gondii infection: parasite invasion and dissemination were slower and mortality was reduced [43].

It has been shown lately that some isoforms of CD209 may influence the development of resistance to infection with paratuberculosis in cows [[44], [45], [46]]. Moreover, it has been reported that overexpression of CD209 in bovine macrophages in vitro leads to the increase of adenovirus infection [47]. Therefore, production of CD209 knock-out is relevant for better understanding of infection mechanisms in cases of paratuberculosis and adenoviruses. The production of such knock-out may potentially enable animals to become resistant to various infections.