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Development of PCR based approach to detect potential mosaicism in porcine embryos
Jongki Cho,Kyungjun Uh,Junghyun Ryu,Xun Fang,Seonggyu Bang,Kiho Lee 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.4
Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.
Development of PCR based approach to detect potential mosaicism in porcine embryos
Cho, Jongki,Uh, Kyungjun,Ryu, Junghyun,Fang, Xun,Bang, Seonggyu,Lee, Kiho The Korean Society of Animal Reproduction and Biot 2020 한국동물생명공학회지 Vol.35 No.4
Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.
Cho, Seongguk,Hong, Riwon,Yim, Poorm,Yeom, Mijung,Lee, Bombi,Yang, Woong Mo,Hong, Jongki,Lee, Hyang Sook,Hahm, Dae–,Hyun Elsevier 2018 Journal of Ethnopharmacology Vol.213 No.-
<P><B>Abstract</B></P> <P><B>Ethnopharmacological relevance</B></P> <P> <I>Schisandra chinensis</I> (Turcz.) Baill (SC), <I>Lycium chinense</I> Mill (LC) and <I>Eucommia ulmoides</I> Oliv (EU) are representative tonic herbal medicines that help to strengthen body muscles and bones making them stronger according to the Donguibogam, a tradition medical book of the Joseon Dynasty in Korea.</P> <P><B>Aim of the study</B></P> <P>To evaluate effects of an herbal formula consisting of SC, LC and EU on muscle atrophy in C2C12 myotubes and in a rat model of immobilization-induced muscle atrophy.</P> <P><B>Materials and methods</B></P> <P>Muscle atrophy was developed by cast immobilization of unilateral hindlimb on rats for 3 weeks. Treatments were administered orally 14 times over 3 weeks. After treatments, we compared the change of body weight, muscle weight, grip strength, muscle fiber size, muscle fiber type shift by Grip strength meter, H&E stain and ATPase stain. And western blot was used for evaluating molecular mechanism in muscle atrophy on C2C12 cells.</P> <P><B>Results</B></P> <P>When taken individually, SC was the most effective of the three in inhibiting tumor necrosis factor alpha (TNF-α)-induced degeneration of C2C12 myogenesis. The formulation with a mass ratio of 2:1:1 SC: LC: EU (SSLE) was more effective against TNF-α-induced muscle atrophy than was a 1:1:1 SC: LC: EU (SLE) formula or any of the single herbal extracts. In a rat model of disuse muscle atrophy, the SSLE formula significantly inhibited reductions in muscle weight, grip strength and muscle fiber size induced by hindlimb immobilization, in a dose-dependent manner. The formula also inhibited immobilization-induced shifting of the muscle fiber type in soleus muscle. Treatment with SSLE inhibited TNF-α-induced expression of the atrogenes atrogin-1 and muscle RING-finger protein 1 in C2C12 cells. The SSLE formula also increased myoblast differentiation markers (myoD and myogenin) and activation of the Akt and mammalian target of rapamycin (mTOR) signaling pathway.</P> <P><B>Conclusion</B></P> <P>These findings suggest that the SSLE formula prevents muscle atrophy through inhibition of the ubiquitin-proteasome system as well as upregulation of myoblast differentiation and muscle protein synthesis in C2C12 cells. Taken together, we conclude that the SSLE formula is invaluable for the development of therapeutic medicines to prevent disuse muscle atrophy and its accompanying muscle weakness.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Formation and Related-Behavior of Micro-bowl Morphology Consisting of Ionic Palladium(II) Complexes
Cho Rong Kim,Chi Won Kim,노태환,이영아,Jongki Hong2,,정옥상 대한화학회 2010 Bulletin of the Korean Chemical Society Vol.31 No.8
Reaction of [(bpy)Pd](PF6)2 (bpy = 2,2'-bipyridine) with racemic bis(isonicotinoyl)-1,1'-bi-2-naphtholate (L) in acetone,and followed by addition of chloroform and solvent evaporation allows to form amorphous micro-bowl morphology consisting of [(bpy)PdL]2(PF6)4 without any template or additive. In contrast, the reaction and recrystallization in acetone for 1 week produce parallel-piped single crystals consisting of [(bpy)3Pd3(μ3-HPO4)2](PF6)2. The formations of micro-bowl and parallel-piped single crystal morphologies appear to be primarily associated with the kinetic and thermodynamic control, respectively. The formation of micro-bowls may be attributed to eruption of organic solvents. Cosolvent effects and chemical properties on the formation of micro-bowl morphology have been observed.