The creation of a multiallele knockout genotype in rabbit using crispr/cas9 and its application in translational medicine

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Somatic mutations of KIT in familial testicular germ cell tumours

Somatic mutations of the KIT gene have been reported in mast cell diseases and gastrointestinal stromal tumours. Recently, they have also been found in mediastinal and testicular germ cell tumours (TGCTs), particularly in cases with bilateral disease. We screened the KIT coding sequence (except exon 1) for germline mutations in 240 pedigrees with two or more cases of TGCT. No germline mutations were found. Exons 10, 11 and 17 of KIT were examined for somatic mutations in 123 TGCT from 93 multiple-case testicular cancer families. Five somatic mutations were identified; four were missense amino acid substitutions in exon 17 and one was a 12bp in-frame deletion in exon 11. Two of seven TGCT from cases with bilateral disease carried KIT mutations compared with 3 out 116 unilateral cases (p = 0.026). The results indicate that somatic KIT mutations are implicated in the development of a minority of familial as well as sporadic TGCT. They also lend support to the hypothesis that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes

Genome-wide linkage screen for testicular germ cell tumour susceptibility loci

A family history of disease is a strong risk factor for testicular germ cell tumour (TGCT). In order to identify the location of putative TGCT susceptibility gene(s) we conducted a linkage search in 237 pedigrees with two or more cases of TGCT. One hundred and seventy-nine pedigrees were evaluated genome-wide with an average inter-marker distance of 10 cM. An additional 58 pedigrees were used to more intensively investigate several genomic regions of interest. Genetic linkage analysis was performed with the ALLEGRO software using two model-based parametric analyses and a non-parametric analysis. Six genomic regions on chromosomes 2p23, 3p12, 3q26, 12p13-q21, 18q21-q23 and Xq27 showed heterogeneity LOD (HLOD) scores of greater than 1, with a maximum HLOD of 1.94 at 3q26. Genome-wide simulation studies indicate that the observed number of HLOD peaks greater than one does not differ significantly from that expected by chance. A TGCT locus at Xq27 has been previously reported. Of the 237 pedigrees examined in this study, 66 were previously unstudied at Xq27, no evidence for linkage to this region was observed in this new pedigree set. Overall, the results indicate that no single major locus can account for the majority of the familial aggregation of TGCT, and suggests that multiple susceptibility loci with weak effects contribute to the diseas

Palynomorphs of the Normapolles group and related plant mesofossils from the Iharkút vertebrate site, Bakony Mountains (Hungary)

Abstract Palynological and paleobotanical investigation of bonebeds and other strata of the Csehbánya Formation from the vertebrate locality at Iharkút (Bakony Mts, Hungary) reveals well-preserved Santonian palynological assemblages dominated by the Normapolles group, with a minor component consisting of other angiosperm pollen, some gymnosperm pollen, and spores. Eleven species of Normapolles-type pollen grains belonging to seven genera and fruit remains of a new taxon, Sphaeracostata barbackae gen. et sp. nov., are described. The new species is very abundant in the material, represented by ca. 1000 specimens. The genus Caryanthus Friis and an unnamed form previously reported from Haţeg by Lindfors et al. (2010) are also present. Plants producing Normapolles-type pollen grains diversified during the Late Cretaceous, with a bloom in the Santonian. The palynostratigraphy of the Upper Cretaceous terrestrial sediments in the studied region is based on Normapolles-related species. The studied assemblage is assigned to the Oculopollis zaklinskaiae-Tetracolporopollenites (Brecolpites) globosus Zone (or Zone C) indicating a late Santonian age. Comparison of the Iharkút palynoflora with other known Upper Cretaceous palynofloras of Central Europe shows diachronous occurrence of Normapolles taxa at different geographic localities and warrants further investigation. The ecological requirements of the amphibian fauna reflect azonal conditions controlled by the availability of water, which is in agreement with the inferred ecological conditions based on the paleobotanical investigations. The fauna is of entirely non-marine character, further supported by isotope studies, in line with our data showing that the palynological samples contain no marine forms

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

Association of genomic domains in BRCA1 and BRCA2 with prostate cancer risk and aggressiveness

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. Weevaluated whether PSVs inBRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 30 region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25-2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63-5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71-4.68; P = 0.00004) and elevated risk of Gleason 8+prostate cancer (HR = 4.95; 95% CI, 2.12-11.54; P = 0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. Significance: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.Peer reviewe