A genome-wide association study of coat color in Chinese Rex rabbits

Coat color is an important phenotypic characteristic of the domestic rabbit (Oryctolagus cuniculus) and has specific economic importance in the Rex rabbit industry. Coat color varies considerably among different populations of rabbits, and several causal genes for this variation have been thoroughly studied. Nevertheless, the candidate genes affecting coat color variation in Chinese Rex rabbits remained to be investigated. In this study, we collected blood samples from 250 Chinese Rex rabbits with six different coat colors. We performed genome sequencing using a restriction site-associated DNA sequencing approach. A total of 91,546 single nucleotide polymorphisms (SNPs), evenly distributed among 21 autosomes, were identified. Genome-wide association studies (GWAS) were performed using a mixed linear model, in which the individual polygenic effect was fitted as a random effect. We detected a total of 24 significant SNPs that were located within a genomic region on chromosome 4 (OCU4). After re-fitting the most significant SNP (OCU4:13,434,448, p = 1.31e-12) as a covariate, another near-significant SNP (OCU4:11,344,946, p = 7.03e-07) was still present. Hence, we conclude that the 2.1-Mb genomic region located between these two significant SNPs is significantly associated with coat color in Chinese Rex rabbits. The well-studied coat-color-associated agouti signaling protein (ASIP) gene is located within this region. Furthermore, low genetic differentiation was also observed among the six coat color varieties. In conclusion, our results confirmed that ASIP is a putative causal gene affecting coat color variation in Chinese Rex rabbits

A novel intramural TGF β 1 hydrogel delivery method to decrease murine abdominal aortic aneurysm and rat aortic pseudoaneurysm formation and progression

Objectives: Aneurysms are generally the result of dilation of all 3 layers of the vessel wall, and pseudoaneurysms are the result of localized extravasation of blood that is contained by surrounding tissue. Since there is still no recommended protocol to decrease aneurysm formation and progression, we hypothesised that intramural delivery of TGF β1 hydrogel can decrease aneurysm and pseudoaneurysm formation and progression. Materials: Male C57BL/6 J mice (12–14 wk), SD rats (200 g) and pig abdominal aortas were used, and hydrogels were fabricated by the interaction of sodium alginate (SA), hyaluronic acid (HA) and CaCO3. Methods: A CaCl2 adventitial incubation model in mice and a decellularized human great saphenous vein patch angioplasty model in rats were used. TGF β1 hydrogel was intramurally delivered after CaCl2 incubation in mice; at day 7, the abdomen in some mice was reopened, and TGF β1 hydrogel was injected intramurally into the aorta. In rats, TGF β1 hydrogel was delivered intramurally after patch angioplasty completion. Tissues were harvested at day 14 and analysed by histology and immunohistochemistry staining. The pig aorta was also intramurally injected with hydrogel. Results: In mice, rhodamine hydrogel was still found between the medium and adventitia at day 14. In the mouse aneurysm model, there was a thicker wall and smaller amount of elastin breaks in the TGF β1 hydrogel-delivered groups both at day 0 and day 7 after CaCl2 incubation, and there were larger numbers of p-smad2- and TAK1-positive cells in the TGF β1 hydrogel-injected groups. In the rat decellularized human saphenous vein patch pseudoaneurysm model, there was a higher incidence of pseudoaneurysm formation when the patch was decellularized using 3% SDS, and delivery of TGF β1 hydrogel could effectively decrease the formation of pseudoaneurysm formation and increase p-smad2 and TAK1 expression. In pig aortas, hydrogels can be delivered between the medium and adventitia easily and successfully. Conclusions: Intramural delivery of TGF β1 hydrogel can effectively decease aneurysm and pseudoaneurysm formation and progression in both mice and rats, and pig aortas can also be successfully intramurally injected with hydrogel. This technique may be a promising drug delivery method and therapeutic choice to decrease aneurysm and pseudoaneurysm formation and progression in the clinic

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells

Abstract Background Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. Methods To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system’s feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. Results We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. Conclusion The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application

Targeted RNA N6‐Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells

Abstract Deficiency of the N6‐methyladenosine (m6A) methyltransferase complex results in global reduction of m6A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6A methylation affects cell fate decisions due to the inability to modulate individual m6A modification in ESCs with precise temporal control. Here, a targeted RNA m6A erasure (TRME) system is developed to achieve site‐specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline‐inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6A site of mRNA and increase mRNA stability with limited off‐target effects. It is further demonstrated that temporal m6A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6A modification in hESCs, enabling cell fate control studies at the epitranscriptional level

Additional file 1: Figure S1. of Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells

showing expression of factors from episomal vectors. a pE3.1 plasmid construction process chart (upper). Schematic representation of seven constructed episomal vectors. pEF1ι EF1ι promoter, pCMV CMV promoter (below). b Quantitative real-time PCR assay for Oct4, Glis1, Klf4, Sox2, L-Myc, linc-RoR, miR-367, miR-302a, miR-302b, miR-302c, and miR-302d. c Western blot assay for Oct4, Glis1, Klf4, Sox2, and L-Myc carried on episomal vectors. GAPDH was used as the loading control. (PDF 171 kb

Additional file 3: Table S2. of Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells

presenting plasmid combinations used for screening low-risk factors. (XLSX 9 kb

Additional file 4: Table S3. of Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells

presenting the method of karyotype analysis. (XLSX 10 kb