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Renyue Wei,Jiawei Lv,Xuechun Li,Yan Li,Qianqian Xu,Junxue Jin,Yu Zhang,Zhonghua Liu 대한수의학회 2020 Journal of Veterinary Science Vol.21 No.1
Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3β inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.
Yongjie Feng,Wei Liu,Dhiraj Kumar,Min Zhu,Renyu Xue,Guangli Cao,Xiaolong Hu,Chengliang Gong 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.4
The receptor tyrosine kinase-like orphan receptor 2 (Ror2) is involved in the Wnt/β-catenin signaling pathway which regulates cell proliferation, polarity, differentiation, migration, metabolism and survival. However, the function of Ror2 in the silkworm Bombyx mori is still undisclosed. In the present investigation, we have made an effort to clone the silkworm Ror2 gene (BmRor2). The sequence analysis showed that the open reading frame (ORF) was 1914 bp in size and encoded a protein with the conserved domains of Ror2 protein. The qRT-PCR results indicated that the BmRor2 gene expression level was the highest in the head among all identified tis sues on 3rd day of the fifth instar larvae. In the gonads of the different development stages, the BmRor2 gene expression level was highest on the 4th day of the fourth instar larvae. The immunofluorescence assay indicated that the BmRor2 protein was located at the cytomembrane. The effects of BmRor2 protein on the expression levels of genes related to TGF-β, Hippo, JAK-STAT and Notch signaling pathways were investigated by qRT-PCR. The expression levels of crumbs (crb), warts (wts), α-catenin (cat), four-jointed (fj), decapentaplegic (dpp), kibra ortholog (kibra), serrate (serr) and c-myc (myc) genes were down-regulated, whereas, suppressor of cytokine signaling 2 (socs 2) gene expression was up-regulated in the cultured BmN cells after the BmRor2 expression level was up-regulated. Further, cell proliferation was demoted and the size of cells was decreased when BmRor2 expression level was elevated. Our current finding recommended that BmRo2 can regulate TGF- β, Hippo, JAKSTAT, and Notch signaling pathways, and affect cell proliferation and size.
Jiawei Lv,Shuang Wu,Renyue Wei,Yan Li,Junxue Jin,Yanshuang Mu,Yu Zhang,Qingran Kong,Xiaogang Weng,Zhonghua Liu 대한수의학회 2019 Journal of Veterinary Science Vol.20 No.3
The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/ Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.