Production of Cloned Pigs Derived from Double Gene Knockout Cells Using CRISPR/Cas9 System and MACS-based Enrichment System

Bumrae Cho,Su Jin Kim,Eun-Jin Lee,Sun Mi Ahn,Jin Seok Lee,Dal-young Ji,Sang Hoon Lee,Jung-Taek Kang 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.4

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of α-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

Production of Cloned Pigs Derived from Double Gene Knockout Cells Using CRISPR/Cas9 System and MACS-based Enrichment System

Cho, Bumrae,Kim, Su Jin,Lee, Eun-Jin,Ahn, Sun Mi,Lee, Jin Seok,Ji, Dal-young,Lee, Sang Hoon,Kang, Jung-Taek The Korean Society of Embryo Transfer 2018 한국동물생명공학회지 Vol.33 No.4

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

Generation of Soluble Human Tumor Necrosis Factor-α Receptor 1-Fc Transgenic Pig

Cho, Bumrae,Koo, Ok Jae,Hwang, Jong-Ik,Kim, Hwajung,Lee, Eun Mi,Hurh, Sunghoon,Park, Sol Ji,Ro, Han,Yang, Jaeseok,Surh, Charles D.,d’Apice, Anthony J.,Lee, Byeong Chun,Ahn, Curie Lippincott Williams Wilkins, Inc. 2011 Transplantation Vol.92 No.2

BACKGROUND.: Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-α (hTNF-α) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs. METHODS.: The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested. RESULTS.: In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-&kgr;B activation in the HEK293-NF-&kgr;B-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-α-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-α. CONCLUSIONS.: We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-α-mediated activation of PECs. Because TNF-α is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation

Cho, Bumrae,Lee, Eun-Jin,Ahn, Sun Mi,Kim, Ghangyong,Lee, Sang Hoon,Ji, Dal-Young,Kang, Jung-Taek Springer-Verlag 2019 Transgenic research Vol.28 No.5

<P> Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the β-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation. </P>

EMB 시스템 시뮬레이션 모델 개발

조범래(Bumrae Cho),김명준(Myoungjune Kim) 한국자동차공학회 2014 한국자동차공학회 부문종합 학술대회 Vol.2014 No.5

EMB (Electro-Mechanical Brake) is an integrated brake module of CBS (Conventional brake system) and active brake (ABS, ESC, Value added) functions. EMB is consist of PMSM which generates braking torque and ball screw which is used to translate rotary motion from PMSM to axial motion of caliper cylinder. The development objective of EMB simulator model is validation of EMB braking control algorithms feasibility. EMB simulator enables to test vehicle braking performance according to properties of EMB HW in the MATLAB/Simulink environment. Therefore, A EMB simulator system is composed of vehicle model, EMB HW model and SW model. A vehicle model by using CarSim simulates the performance of vehicles in response to driver braking control. EMB HW model by using system identification is built by mathematical models from measure data. It is a simulation validated by comparing simulation results with vehicle test results.

4W BBW 시스템의 CBS/ABS 제어

조범래(Bumrae Cho),정지현(Myo),김명준(MyoungJune Kim) 한국자동차공학회 2012 한국자동차공학회 부문종합 학술대회 Vol.2012 No.5

BBW system can be used for CBS/ABS in this paper. The proposed BBW can be applied in 4W EMB which is Front/Rear EMB actuator. The proposed BBW system has 3 part advantages such as environment friendly, control and packaging. The proposed BBW system is controlled by EMB typical pedal to compare hydraulic brake system. From this research, we have obtained BBW CBS and ABS feasibility.

[Technical Paper] Fault Tolerant Control of the PMSM Drive for Electro-Hydraulic Brake Systems

Seungjin Yoo,Bumrae Cho,Seung-Han You 한국자동차공학회 2019 한국 자동차공학회논문집 Vol.27 No.11

This paper presents a fault-tolerant control algorithm for electro-hydraulic brake systems that use the permanent magnet synchronous motor(PMSM) as a brake booster. In order to deal with motor current sensor faults, an observer-based feedback controller is proposed. Based on the voltage equation defined on the synchronous reference frame, the observer estimates the motor current with the projection matrix filtering out the abnormal sensor signals and its estimated counterparts. In order to improve accuracy, the observed current is transformed into phase current and fed into the input voltage of the observer-based feedback controller to compensate for the inverter dead-time. In case both the position sensor and the current sensor are faulty, the motor current is controlled by the open-loop algorithm. The open-loop current controller is designed based on the assumption that the rotor magnet is aligned sufficiently well with the current vector. The premise is satisfied by designing a position sensorless brake pressure controller in such a way that the magnitude of the current vector is determined from the target brake pressure and the target speed is derived from the pressure control error. The experimental result shows that the current estimation error is less than 10 % and approximately 50 % of the maximum braking pressure can still be generated even if the current and position sensors are all faulty.

Advenced research for generation of genetically engineered pigs

Jung-Taek Kang,Bumrae Cho,Eun-Jin Lee,Sun-Mi Ahn,Su-Jin Kim,Yun-Jin Yoon,Jin-Seok Lee,Dal-Young Ji,Shin-Hye Ahn,Sang-Hoon Lee 한국실험동물학회 2017 한국실험동물학회 학술발표대회 논문집 Vol.2017 No.2

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide

Park, Sol Ji,Cho, Bumrae,Koo, Ok Jae,Kim, Hwajung,Kang, Jung Taek,Hurh, Sunghoon,Kim, Su Jin,Yeom, Hye Jung,Moon, Joonho,Lee, Eun Mi,Choi, Ji Yei,Hong, Ju Ho,Jang, Goo,Hwang, Joing-Ik,Yang, Jaeseok,Le Springer-Verlag 2014 Transgenic research Vol.23 No.3

ZTA 제조시 알루미나 입자크기가 치밀화 거동에 미치는 영향

채지훈,조범래,Chae, Jihoon,Cho, Bumrae 한국재료학회 2013 한국재료학회지 Vol.23 No.4

In order to increase the toughness of ZTA(zirconia toughened alumina) ceramics, the present study focused on rearrangement and densification of particles according to the particle size of the parent material. When rough alumina was used for production of ZTA, densification behavior was observed in the specimen sintered at a temperature over $1550^{\circ}C$. However, it was found that the densification behavior was occurred in the specimen sintered at $1450^{\circ}C$ when fine alumina powder was used. High relative density exceeding 98% was obtained when fine alumina powder was mixed with 15 wt% of 3Y-TZP and sintered at $1600^{\circ}C$. Also, a hardness of 1820.2 Hv was obtained when a specimen containing 10 wt% of 3Y-TZP was sintered at $1600^{\circ}C$. In the case of 3Y-TZP containing rough alumina powder that had been sintered the hardness value was around 1720.3 Hv. It was predicted that an improved toughening effect in ZTA could be achieved by using finer alumina powder as the parent material.