Mitochondrial genome plasticity of mammalian species.

There is an ongoing process in which mitochondrial sequences are being integrated into the nuclear genome. The importance of these sequences has already been revealed in cancer biology, forensic, phylogenetic studies and in the evolution of the eukaryotic genetic information. Human and numerous model organisms' genomes were described from those sequences point of view. Furthermore, recent studies were published on the patterns of these nuclear localised mitochondrial sequences in different taxa.However, the results of the previously released studies are difficult to compare due to the lack of standardised methods and/or using few numbers of genomes. Therefore, in this paper our primary goal is to establish a uniform mining pipeline to explore these nuclear localised mitochondrial sequences.Our results show that the frequency of several repetitive elements is higher in the flanking regions of these sequences than expected. A machine learning model reveals that the flanking regions' repetitive elements and different structural characteristics are highly influential during the integration process.In this paper, we introduce a general mining pipeline for all mammalian genomes. The workflow is publicly available and is believed to serve as a validated baseline for future research in this field. We confirm the widespread opinion, on - as to our current knowledge - the largest dataset, that structural circumstances and events corresponding to repetitive elements are highly significant. An accurate model has also been trained to predict these sequences and their corresponding flanking regions.

Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal.

Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.

Nuclear mitochondrial DNA sequences in the rabbit genome.

Numtogenesis is observable in the mammalian genomes resulting in the integration of mitochondrial segments into the nuclear genomes (numts). To identify numts in rabbit, we aligned mitochondrial and nuclear genomes. Alignment significance threshold was calculated and individual characteristics of numts were analysed. We found 153 numts in the nuclear genome. The GC content of numts were significantly lower than the GC content of their genomic flanking regions or the genome itself. The frequency of three mammalian-wide interspersed repeats were increased in the proximity of numts. The decreased GC content around numts strengthen the theory which supposes a link between DNA structural instability and numt integration.

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use.

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality.

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.

Glomerulosclerosis in transgenic rabbits with ubiquitous Venus protein expression.

Focal segmental glomerulosclerosis (FSGS) is a potential cause of nephrotic syndrome both in humans and pet mammals. Glomerulopathy was reported earlier in green fluorescent protein (GFP) transgenic (TG) mice, but glomerulosclerosis has not been examined in GFP TG rabbits so far. In the present study, the potential manifestation of FSGS was investigated in both Venus TG rabbits generated by Sleeping Beauty (SB) transposition and age-matched control New Zealand White (NZW) rabbits. Venus protein fluorescence was detected by confocal microscopy and quantified by microplate reader. Urinalysis, haematology, serum biochemistry and renal histology were performed to assess the signs of FSGS. Higher levels of Venus fluorescence were determined in renal cortex samples than in the myocardium by both methods. Urinalysis revealed proteinuria in Venus heterozygote TG bucks, while Venus homozygote TG bucks developed microscopic haematuria. Supporting the urinalysis data, the histological findings of FSGS (glomerulomegaly and sclerotic glomeruli) were observed in renal cortex sections of Venus TG rabbits. Taken together, Venus TG bucks were diagnosed with FSGS; thus, this type of glomerulopathy could be a common disease in TG animals overexpressing GFP.

Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

Placenta-specific gene manipulation in rabbits.

Lentiviral gene constructs can be efficiently and specifically delivered to trophoblast cell lineages in rodents. In vivo genetic manipulation of trophoblast cell lines enables functional and developmental studies in the placenta. In this report we show that genetic modification can be produced in the extraembryonic tissues of rabbits by lentiviral gene constructs. When 8-16 cell stage embryos were injected with lentiviral particles, strong reporter gene expression resulted in the rabbit placenta. The expression pattern displayed some mosaicism. A strikingly high degree of mosaic GFP expression was detected in some parts of the yolk sac, which is a hypoblast-derived tissue. Whereas expression of the reporter gene construct was detected in placentas and yolk sacs, fetuses never expressed the transgene. As rabbits are an ideal model for functional studies in the placenta, our method would open new possibilities in rabbit biotechnology and placentation studies.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.

A genomic landscape of mitochondrial DNA insertions in the pig nuclear genome provides evolutionary signatures of interspecies admixture.

Nuclear DNA sequences of mitochondrial origin (numts) are derived by insertion of mitochondrial DNA (mtDNA), into the nuclear genome. In this study, we provide, for the first time, a genome picture of numts inserted in the pig nuclear genome. The Sus scrofa reference nuclear genome (Sscrofa10.2) was aligned with circularized and consensus mtDNA sequences using LAST software. A total of 430 numt sequences that may represent 246 different numt integration events (57 numt regions determined by at least two numt sequences and 189 singletons) were identified, covering about 0.0078% of the nuclear genome. Numt integration events were correlated (0.99) to the chromosome length. The longest numt sequence (about 11 kbp) was located on SSC2. Six numts were sequenced and PCR amplified in pigs of European commercial and local pig breeds, of the Chinese Meishan breed and in European wild boars. Three of them were polymorphic for the presence or absence of the insertion. Surprisingly, the estimated age of insertion of two of the three polymorphic numts was more ancient than that of the speciation time of the Sus scrofa, supporting that these polymorphic sites were originated from interspecies admixture that contributed to shape the pig genome.

Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector.

Efficient production of transgenic animals using low-titer lentiviral constructs remains challenging. Here we demonstrate that microinjection of simian immundeficiency virus-derived lentiviral constructs can produce transgenic mice and rats with high efficiency even when using low-titer virus preparations.

Characterization of the rabbit neonatal Fc receptor (FcRn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses FcRn.

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits--having one extra copy of the FcRn when hemizygous and two extra copies when homozygous--showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.