Non­viral transfection methods optimized for miRNA delivery to human dermal fibroblasts.

Fibroblasts are beneficial model cells for in vitro studies and are frequently used in tissue engineering. A number of transfection reagents have been employed to deliver microRNAs (miRNAs/miRs) into cells for genetic manipulation. The present study aimed to establish an effective method of transient miRNA mimic transfection into human dermal fibroblasts. The experimental conditions included three different methods: Physical/mechanical nucleofection, and two lipid­based methods, Viromer® Blue and INTERFERin®. To evaluate the impact of these methods, cell viability and cytotoxicity assays were performed. The silencing effect of miR­302b­3p was revealed to alter the expression levels of its target gene carnitine O­octanoyltransferase (CROT) by reverse transcription­quantitative PCR. The present study showed that all selected non­viral transient transfection systems exhibited good efficiency. It was also confirmed that nucleofection, for which a 21.4­fold decrease in the expression of the CROT gene was observed 4 h after 50 nM hsa­miR­302b­3p transfection, was the most effective method. However, these results indicated that lipid­based reagents can maintain the silencing effect of miRNAs up to 72 h after transfection. In summary, these results indicated that nucleofection may be the optimal method for the transport of small miRNA mimics. However, lipid­based methods allow for the use of lower concentrations of miRNA and maintain longer­lasting effects.

Characteristics of diluted-stored and post-thawed semen of Hutsul stallions.

The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, using a CASA system. There were no differences in sperm motility (P = 0.3372) or morphology between the groups, although the progressive motility was higher in thawed semen (P = 0.0151), while the sperm vitality was higher in diluted-stored semen (P = 0.00517). This study demonstrates that semen from Hutsul horses is suitable for cryopreservation, thus supporting the creation of a sperm bank as a genetic reserve for representatives of this breed.

Current standards and pitfalls associated with the transfection of primary fibroblast cells.

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.

The perspective of the incompatible of nucleus and mitochondria in interspecies somatic cell nuclear transfer for endangered species.

Taking into account the latest Red List of the International Union for Conservation of Nature in which 25% of all mammals are threatened with extinction, somatic cell nuclear transfer (SCNT) could be a beneficial tool and holds a lot of potential for aiding the conservation of endangered, exotic or even extinct animal species if somatic cells of such animals are available. In the case of shortage and sparse amount of wild animal oocytes, interspecies somatic cell nuclear transfer (iSCNT), where the recipient ooplasm and donor nucleus are derived from different species, is the alternative SCNT technique. The successful application of iSCNT, resulting in the production of live offspring, was confirmed in several combination of closely related species. When nucleus donor cells and recipient oocytes have been used in many other combinations, very often with a very distant taxonomical relation iSCNT resulted only in the very early stages of cloned embryo development. Problems encountered during iSCNT related to mitochondrial DNA (mtDNA)/genomic DNA incompatibility, mtDNA heteroplasmy, embryonic genome activation of the donor nucleus by the recipient oocyte and availability of suitable foster mothers for iSCNT embryos. Implementing assisted reproductive technologies, including iSCNT, to conservation programmes also raises concerns that the production of genetically identical populations might cause problems with inbreeding. The article aims at presenting achievements, limitations and perspectives of iSCNT in maintaining animal biodiversity.