Application of transposon systems in the transgenesis of bovine somatic and germ cells

Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type

Interleukin-10 Polymorphisms in Association with Prognosis in Patients with B-Cell Lymphoma Treated by R-CHOP

Interleukin-10 (IL10) plays an important role in initiating and maintaining an appropriate immune response to non-Hodgkin lymphoma (NHL). Previous studies have revealed that the transcription of IL10 mRNA and its protein expression may be infl uenced by several single-nucleotide polymorphisms in the promoter and intron regions, including rs1800896, rs1800871, and rs1800872. However, the impact of polymorphisms of the IL10 gene on NHL prognosis has not been fully elucidated. Here, we investigated the association between IL10 polymorphisms and NHL prognosis. This study involved 112 NHL patients treated at the National Cancer Center, Korea. The median age was 57 years, and 70 patients (62.5%) were men. Clinical characteristics, including age, performance status, stage, and extra-nodal involvement, as well as cell lineage and International Prognostic Index (IPI), were evaluated. A total of four polymorphisms in IL10 with heterozygous alleles were analyzed for hazard ratios of overall survival (OS) and progression-free survival (PFS) using Cox proportional hazards regression analysis. Diffuse large B-cell lymphoma was the most common histologic type (n = 83), followed by T-cell lymphoma (n = 18), mantle cell lymphoma (n = 6), and others (n = 5). Cell lineage, IPI, and extra-nodal involvement were predictors of prognosis. In the additive genetic model results for each IL10 polymorphism, the rs1800871 and rs1800872 polymorphisms represented a marginal association with OS (p = 0.09 and p = 0.06) and PFS (p = 0.05 and p = 0.08) in B-cell lymphoma patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These findings suggest that IL10 polymorphisms might be prognostic indicators for patients with B-cell NHL treated with R-CHOP

Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation

Background Genome editing has been considered as powerful tool in agricultural fields. However, genome editing progress in cattle has not been fast as in other mammal species, for some disadvantages including long gestational periods, single pregnancy, and high raising cost. Furthermore, technically demanding methods such as microinjection and somatic cell nuclear transfer (SCNT) are needed for gene editing in cattle. In this point of view, electroporation in embryos has been risen as an alternative. Results First, editing efficiency of our electroporation methods were tested for embryos. Presence of mutation on embryo was confirmed by T7E1 assay. With first combination, mutation rates for MSTN and PRNP were 57.6% ± 13.7% and 54.6% ± 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% ± 23.6% for MSTN, 84.5% ± 18.0% for BLG. Afterwards, the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing. Thirteen recipients were transferred for MSTN/PRNP, 4 calves were delivered, and one calf underwent an induction for double KO. Ten surrogates were given double-KO embryos for MSTN/BLG, and four of the six calves that were born had mutations in both genes. Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied. Finally, MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.This study was financially supported by the National Research Foundation of Korea (NRF-2021R1A5A1033157 for SRC program: 382 Comparative medicine Disease Research Center; NRF-2021R1F1A105195313), the Research Institute of Veterinary Science, the BK21 Four for Future Veterinary Medicine Leading Education and Research Center, and a Seoul National University (SNU) grant (#550e2020005

Acaulosproa koreana, a New Species of Arbuscular Mycorrhizal Fungi (Glomeromycota) Associated with Roots of Woody Plants in Korea

A new species of arbuscular mycorrhizal fungi (Glomeromycota), Acaulospora koreana, was isolated from forest soils in South Korea. This novel fungus was collected from the rhizosphere of Lindera obtusiloba and Styrax obassia in forest and propagated with Sorghum bicolor in pot. Morphological characteristics of spores of A. koreana are rarely distinguished from Acaulospora mellea, which is reported as one of the most abundant mycorrhizal species in Korea. However, molecular evidence of rDNA sequence using improved primers for glomeromycotan fungal identification strongly supported that A. koreana is different from A. mellea but also any other species belonging to the genus Acaulospora. This is the first novel glomeromycatan fungus introduced in South Korea, but it suggests that there is a high possibility for discovering new arbuscular mycorrhizal fungi considering the abundance of plant species and advanced phylogenetic analysis technique

Dosimetric and radiobiological comparison in different dose calculation grid sizes between Acuros XB and anisotropic analytical algorithm for prostate VMAT.

To investigate feasible treatment planning parameters, we aimed to evaluate the dosimetric and radiobiological impact of the dose calculation algorithm and grid size in the volumetric modulated arc therapy (VMAT) plan for prostate cancer. Twenty patients were selected, and the treatment plans were initially generated with anisotropic analytical algorithm (AAA) and recalculated with Acuros XB (AXB) algorithm. Various dose grids were used for AXB (1, 2, and 3 mm) and AAA (1, 3, and 5 mm) plan. Dosimetric parameters such as homogeneity index (HI) and conformity index (CI), and radiobiological parameters such as tumor control probability (TCP) and normal tissue complication probability (NTCP) were calculated. Significant differences were observed in the planning target volume (PTV) coverage between both algorithms, and the V95%, HI, and CI of AAA were significantly affected by grid (p < 0.01). On 1 mm grid, the mean rectal dose difference between both algorithms was 2.87% of the prescription dose (p < 0.01), which was the highest among the critical organs. The TCP and NTCP of the AAA were higher than those of AXB (p < 0.01). Compared to AXB with 1 mm grid, the 2 mm grid showed comparable dose calculation accuracy with short calculation time. This study found that the PTV and rectum show significant differences according to dose calculation algorithm and grid. Considering the dose calculation performance for heterogeneous area, we recommend AXB with 2 mm grid for improving treatment efficiency of prostate VMAT