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The Relative Abundances of Human Leukocyte Antigen-E, α-Galactosidase A and α-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells
The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from hFUT2, hGLA and HLA-E transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants. Furthermore, although TSA-based epigenomic modulation has given rise to a remarkable increase in the expression levels of Galα1→3Gal (α-Gal) epitopes that have been determined by lectin blotting analysis, their semi-quantitative profiles have dwindled profoundly in both TSA-exposed and unexposed 3×TG ACFCs as compared to their nTG counterparts. In conclusion, thoroughly exploring proteomic signatures in such epigenetically modulated ex vivo models devised on hFUT2×hGLA×HLA-E triple-transgenic ACFCs that display augmented reprogrammability of translational activities of two mRNA transcripts coding for rhα-Gal A and HLA-E proteins might provide a completely novel and powerful research tool for the panel of further studies. The objective of these future studies should be to multiply the tri-transgenic pigs with the aid of somatic cell nuclear transfer (SCNT)-based cloning for the purposes of both xenografting the porcine cutaneous bioprostheses and dermoplasty-mediated surgical treatments in human patients.
The applicability of nanobiotechnology-related approaches to veterinary medicine and assisted animal reproduction – A review
Abstract The development and optimization of nanobiotechnology has recently contributed to the elaboration of a wide spectrum of nanoparticlebased strategies that are reliable and feasible for a broad panel of practical applications in different disciplines of biological, agricultural, nutritional, biopharmaceutical, and biomedical research fields. Taking into account the aforementioned facts, the primary goal of this article is to provide an insightful interpretation of not only the topical and coming trends, but also the research highlights related to devising less time- and labor-consuming processes, less cytotoxic procedures, and the most – relatively speaking – effective and inter-disciplinary nano-applications that are being implemented in veterinary medicine, reproductive biotechnology, and their combinations with livestock breeding and animal production.
A Comprehensive Look at the -13910 C>T LCT Gene Polymorphism as a Molecular Marker for Vitamin D and Calcium Levels in Young Adults in Central and Eastern Europe: A Preliminary Study
Intolerance to dairy products resulting from the abnormal digestion of milk sugar (lactose) is a common cause of human gastrointestinal disorders. The aim of this study was to show that the -13910 C>T LCT gene polymorphism, together with genotypes of selected VDR gene polymorphisms and diet and nutritional status parameters, can impact the prevalence of vitamin D and calcium deficiency in young adults. This study was conducted on a group of 63 people, which comprised 21 individuals with primary adult lactase deficiency, and a control group of 42 individuals with no hypolactasia. The LCT and VDR gene genotypes were assessed using PCR restriction fragment length polymorphism (PCR-RFLP) analysis. A validated HPLC method was used to determine serum concentrations of 25(OH)D2 and 25(OH)D3. Atomic absorption spectrometry was used to determine calcium levels. Their diets (self-reported 7-day estimated food record), estimated calcium intakes based on the ADOS-Ca questionnaire and basic anthropometric parameters were assessed. The CC genotype associated with hypolactasia was found in 33.3% of the subjects. The presence of the CC variant of the LCT gene polymorphism in the study group of young Polish adults was found to be associated with significantly lower milk (134.7 ± 66.7 g/d vs. 342.5 ± 176 g/d; p = 0.012) and dairy product consumption (78.50 ± 36.2 g/d vs. 216.3 ± 102 g/d; p = 0.008) compared with lactase persistence. At the same time, people with adult-type primary intolerance were found to have statistically significant lower serum levels of vitamin D and calcium (p < 0.05). There was a higher chance of vitamin D and calcium deficiency and a lower intake in the group exhibiting lactase non-persistence (OR > 1). The AA variant of the VDR gene’s BsmI polymorphism present in people with hypolactasia may further contribute to an increased risk of vitamin D deficiency. Exclusion of lactose from the diet, combined with impaired vitamin D metabolism, may also lead to inhibited calcium absorption by the body. Further research should be carried out on a larger group of subjects to clarify the relationship between lactase activity and vitamin D and calcium levels in young adults.
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.