http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Establishment of CRISPR/Cpf1 vector transfected cell line
Seon-Ung Hwang,Mirae Kim,Sang-Hwan Hyun 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05
Severe combined immune deficiency (SCID) pig is very important research model for biomedical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted cancer or stem cell of human origin. FOXN1 gene encodes a transcription factor essential for the development and function of thymic epithelial cells (TECs), the primary lymphoid organ that supports T-cell development and selection. In this study, we are going to produce the FOXN1 KO SCID pigs using the Crispr/Cpf1 method. Porcine genomic DNA sequences were analyzed and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed crDNA oligos was synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). To generate the AsCpf1-mCherry-Puro construct, pTE4396 (#74041; Addgene, Cambridge, MA, USA) was modified by removing the NeoR/KanR sequence using BstBI and SmaI. Then, the mCherry-Puro sequence from pSicoR-Ef1a-mCh-Puro (#31845; Addgene, Cambridge, MA, USA) digested with the same restriction enzymes was inserted into the aforementioned NeoR/KanR-deleted vector. The crDNA #1 or crDNA #2 was inserted into the pTE4396 and AsCpf1-mCherry-Puro vectors in the U6 promoter region using BsmBI enzyme, respectively. The two vectors were transfected with lipofectamine 3000 (Life Technologies, Grand Island, NY, USA) and selected with puromycin and G-418 antibiotics. As a result, we established a cell line into which two vectors (pTE4396+crFOXN1#2 and AsCpf1- mCherry-Puro+ crFOXN1#1) and were inserted. Further studies are needed to characterize FOXN1 KO cell lines.
Production of FOXN1 Knockout Porcine Embryos Via Microinjection of CRISPR/Cpf1 mRNA
Seon-Ung Hwang,Mirae Kim,Sang-Hwan Hyun 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
Severe combined immune deficiency (SCID) pig is the important animal model for translational medical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted human cancer or stem cells. FOXN1 gene encodes a transcription factor essential for the development and function of thymic epithelial cells (TECs), the primary lymphoid organ that supports T-cell development and selection. This study was performed to produce the FOXN1 knockout (KO) SCID pigs’ embryos using the Crispr/Cpf1 system. Porcine genomic DNA sequences were analyzed, and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed T7-crDNA oligos were synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). pTE4396 (#74- 041; Addgene, Cambridge, MA, USA) vector was used as a template for AsCpf1 RNA synthesis. MEGAshortscript™ T7 Transcription Kit (Ambion, Austin, TX, USA) was used for the pFOXN1 target-crRNA synthesis, and RiboMAX™ Large Scale RNA Production Systems (Promega, Madison, WI) was used for the AsCpf1 RNA synthesis. The RNAs were diluted in microinjection buffer (10 mM Tris-HCl and 1 mM EDTA). 50 ng/μL of each pFOXN1 target-crRNA and 100 ng/μL AsCpf1 mRNA were co-injected into the cytoplasm of pronuclear stage PA embryos. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean±SEM. As a result, the cleavage rates were no significant (p<0.05) difference between the control and microinjected groups (79.0±6.3 and 67.9±4.4), and there was no significant (p<0.05) difference in the development rate of blastocysts (60.6±12.1 and 49.3±6.6). These results suggest that there is no negative effect of embryo development by microinjection. To further analyze gene editing efficiency, it will be required to assay Indel and sequencing in the gDNA of a single blastocyst.
Hwang, Seon-Ung,Kim, Kyu-Jun,Kim, Eunhye,Yoon, Junchul David,Park, Kyu Mi,Jin, Minghui,Han, Yongquan,Kim, Mirae,Lee, Gabsang,Hyun, Sang-Hwan Elsevier 2018 Theriogenology Vol.113 No.-
<P><B>Abstract</B></P> <P>Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during <I>in vitro</I> maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and <I>in vitro</I> fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 μM). After 42 h of IVM, the 30 μM LPA-treated group showed a significant (<I>P</I> <<I>0.05</I>) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 μM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 μM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 μM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen<I>, PCNA</I>) in the oocytes and cumulus cells of the individuals in the 30 μM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (<I>uPAR</I>) in cumulus cells was significantly increased in the 30 μM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 μM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.</P>