pHOxsFV벡터와 배아주간세포를 이용한 형질전환생쥐 생산 기초연구

이훈택,이봄이,정길생,김진회 건국대학교 동물자원연구센터 1998 動物資源硏究誌 Vol.19 No.-

pHook™-1 hapten 4-ethoxy-methylene-2-phenyl-2-oxazolin-5one(phOX)의 단일 항체 sFV를 암호화 하고 있으며, murine의 Ig k-chain V-J2-C 영역유래의 signal peptide에 의하여 항체를 세포 표면에 배열시키도록 고안되어 있다. 또한, 항체를 세포막 바깥쪽에 부착되어 있도록 하기 위해 PDGFR 유래의 transembrane domain의 C 말단에 결합되어 있다. 이렇게 고안된 vector을 발현하는 세포는 세포막에 sFV을 발현함으로, phOX로 코팅된 자석베드를 이용하여 배양체로부터 목적의 유전자를 발현하는 세포만을 분리할 수 있을 것이다. 따라서, 본 연구는 pHook™-1 유전자를 co-transfection함으로써 목적의 유전자를 가진 배아주간세포를 단시간 내에 효율적으로 선발하기 위하여 실시하였다. 또한, 배아주간세포에서 목적의 DNA 발현 또는 존재를 검증하기 위해 DNA 발현 또는 존재를 검증하기 위해 PCR 방법과 조직화학적 방법을 사용하였다. 형질전환유전자 발현을 transfection(유전자 전이) 후 4∼14일 사이에 모든 배아주간세포에서 확인되었다. Magnetic bead를 이용하여 선발된 세포에서 co-transfected DNA는 배아주간세포에서 효율절으로 삽입되었으며, 선발된 세포의 약 90%는 co-transfected 유전자를 발현하였다. 이 결과는 세포생리학에서 특이 유전자의 급성변이와 만성변이를 연구하거나, 또는 형질전환동물을 생산하기 위해 pHook™-1 목적유전자와 함께 전이함으로서 효율적으로 목적의 유전자를 가진 세포를 선발 가능함으로써 보다 간편하게 형질전환도 동물의 생산에 이용 가능하다는 사실을 확인하였다. pHook™-1 encodes a single-chain antibody(sFv) directed toward the hapten 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one(phOx): the signal peptide from the murine Igk-chain V-J2-C region is fused in front of coding region of the sFv to direct the antibody to the plasma membrane. The antibody is fused at the C-termius to the transmembrane domain from the platelet derived growth factor receptor(PDGFR), allowing the antibody to be anchored and displayed on the extracellular side of theemmbrane. Transfected cells expression sFv can be isolated from whole cultures by using magnetic coated with phOx and a strong magnetic strand. Thus, the present study was designed to apply the embryonic stem cells by using pHook™-1 . Cell-transduction efficiency was measured by morphometric analysis. Polymerase chain reaction and histochemistry were used to detect the presence and/or expression of objective DNA in embryonic stem cells. Transgene expression was detected in all cases between 4 and 14 day after transfection. In selected cells using magnetic bead, co-transfected DNA was also incorporated efficiently in embryonic stem cells and approximately 90% of the selected cells expressed co-transfected gene. This result suggested that this selection system can be used as a feasible tool, when pHook™-1 is cotransfected with objective gene, to isolate and study for acute and chronic changes of a specific gene in cellular physiology.

Construction of Global Cohesin Marker for Cellulosome from Clostridia spp with Cross-Species Cohesin-Dockerin Interaction

Bom Yi PARK,Sang Duck JEON,Su Jung KIM,Jeong Eun HYEON,So heyong KIM,Sung Ok HAN 한국생물공학회 2015 한국생물공학회 학술대회 Vol.2015 No.10

Three cases of rare SRY-negative 46,XX testicular disorder of sexual development with complete masculinization and a review of the literature

Bom Yi Lee,Shin Young Lee,Yeon Woo Lee,Shin Young Kim,Jin Woo Kim,Hyun Mee Ryu,Joong Shik Lee,So Yeon Park,Ju Tae Seo 대한의학유전학회 2016 대한의학유전학회지 Vol.13 No.2

Purpose: To identify the clinical characteristics of SRY-negative male patients and genes related to male sex reversal, we performed a retrospective study using cases of 46,XX testicular disorders of sex development with a review of the literature. Materials and Methods: SRY-negative cases of 46,XX testicular disorders of sex development referred for cytogenetic analysis from 1983 to 2013 were examined using clinical findings, seminal analyses, basal hormone profiles, conventional cytogenetic analysis and polymerase chain reaction. Results: Chromosome analysis of cultured peripheral blood cells of 8,386 individuals found 19 cases (0.23%) with 46,XX testicular disorders of sex development. The SRY gene was confirmed to be absent in three of these 19 cases (15.8%). Conclusion: We report three rare cases of SRY-negative 46,XX testicular disorders of sex development. Genes on autosomes and the X chromosome that may have a role in sex determination were deduced through a literature review. These genes, through differences in gene dosage variation, may have a role in sex reversal in the absence of SRY.

Prenatally Diagnosed Uncommon Mosaic Autosomal Trisomy

Bom-Yi Lee,So-Yeon Park,Moon-Hee Lee,Jin-Woo Kim,Ju-Yeon Park,Eun-Young Choi,Yeon-Woo Lee,Ah-Rum Oh,Shin-Young Lee,Min-Hyung Kim,Hyun-Mee Ryu 대한의학유전학회 2009 대한의학유전학회지 Vol.6 No.1

Prenatal diagnosis of rare autosome mosaicism involvingchromosomes other than chromosome 13, 18, 21 or the sex chromosome is encountered prognostic dilemma during genetic counseling. We report four cases of level Ⅲ uncommon mosaicism of trisomy 5, 16 and 20,diagnosed prenatally. In case 1 with mosaic trisomy 20, there was a higher mosaic ratio of trisomy 20 in the repeat amniocentesis (62.1%) than in the first (36.6%) with normal fetal ultrasound finding except for a relatively small aorta on a 3-vessel view of the fetal heart. Case 2 showed a low rate of mosaic trisomy 20 (5.25%) in cultured amniocytes but normal karyotype in the repeat amniocentesis, who delivered a normal healthy baby. Case 3 showed a 13.6% of trisomy 16 mosaicism in the 30 cells of cultured amniocytes. Sixty cells from a fetal blood sample at termination showed non-mosaic 46,XX normal karyotype, while skin fibroblasts had 22.5% trisomy 16 in 40 metaphases. The autopsy showed ventricular septal defect (VSD). Case 4 with low grade mosaicism (10.5%) of trisomy 5 resulted in elective termination, though the ultrasoumd showed growsly normal fetus. Although level Ⅲ mosaicism is regarded as true mosaicism, it is difficult to predict the outcome of the fetus with rare mosaic autosome trisomy. Therefore mosaic autosome trisomy of fetus should be carefully interpreted with more various approaches including repeat sampling and targeted fetal ultrasound. 산전에서 성염색체와 13번, 18번, 21번 염색체를 제외한상 염색체의 모자이시즘은 발생빈도가 낮고 증례보고가 적어서 예후 예측이 어렵다. 저자들은 삼염색체성 5번, 16번, 20번의 산전진단 4례를 보고하고자 한다. 모자잌 삼염색체성 20번 2례 중 증례 1은 양수 염색체 검사에서 36.6%의 모자이시즘을 보였으나 재검한 양수 검사에서는 보다 높은 빈도 (62.1%)를 보였다. 증례 2에서는 양수 염색체 검사에서 모자이시즘 삼염색체성 20번이 5.25% 였으나, 재검 양수천자결과는 정상 핵형을 보였다. 증례 3은 30개의 양수세포에서 삼염색체성 16번의 모자이시즘이 13.6% 관찰되었다. 임신 종결 후, 총 60개의 태아 혈액 세포에서 모자이시즘 없는 정상 핵형이 관찰되었으나 태아의 피부 섬유아세포에서 얻은 40개의 중기상 세포에서는 22.5%의 삼염색체성 16번 모자이시즘을 보였다. 부검결과 심실중격결손(ventricular septal defect)이 관찰되었다. 증례 4는 76개의 중기상 세포에서 10.5%의 삼염색체성 5번 모자이시즘을 보였으나 태아의 초음파검사에서는 정상소견을 보였다. Level Ⅲ 모자이시즘은 진성 모자이시즘으로 간주되지만 발생빈도가 낮은 상염색체의 삼염색체성 모자이시즘은 태아의 예후를 예견하기 어려우므로 산전 진단시 여러 조직의 재검 및 태아 초음파 소견과 함께 다양한 임상적 접근 방법으로 그 해석에 신중을 기해야 할 것으로 사료된다.

An unusual de novo duplication 10p/deletion 10q syndrome

Bom-Yi Lee,Ju-Yeon Park,Yeon-Woo Lee,Ah-Rum Oh,Shin-Young Lee,Eun-Young Choi,Moon-Young Kim,Hyun-Mee Ryu,So-Yeon Park 대한의학유전학회 2015 대한의학유전학회지 Vol.12 No.1

We herein report an analysis of a female baby with a de novo dup(10p)/del(10q) chromosomal aberration. A prenatal cytogenetic analysis was performed owing to abnormal ultrasound findings including a choroid plexus cyst, prominent cisterna magna, and a slightly medially displaced stomach. The fetal karyotype showed additional material attached to the terminal region of chromosome 10q. Parental karyotypes were both normal. At birth, the baby showed hypotonia, upslanting palpebral fissures, a nodular back mass, respiratory distress, neonatal jaundice and a suspicious polycystic kidney. We ascertained that the karyotype of the baby was 46,XX,der(10)(pter→q26.3::p11.2→pter) by cytogenetic and molecular cytogenetic analyses including high resolution GTG-and RBG-banding, fluorescence in situ hybridization, comparative genomic hybridization, and short tandem repeat marker analyses. While almost all reported cases of 10p duplication originated from one of the parents with a pericentric inversion, our case is extraordinarily rare as the de novo dup(10p)/ del(10q) presumably originated from a rearrangement at the premeiotic stage of the parental germ cell or from parental germline mosaicism.

Smart AAO Membrane for Multiple Gating System

Bom-yi Lee,Jin Kon Kim 한국고분자학회 2016 한국고분자학회 학술대회 연구논문 초록집 Vol.41 No.2

Follow - up of foreign DNA by lipofectin - aided sperm - mediated gene transfer

Bom Yi Lee,Jin Hoi Kim,Hoon Taek Lee,Kil Saeng Chung 한국동물자원과학회(구 한국축산학회) 1996 한국축산학회 심포지움자료집 Vol.- No.-

Prenatal diagnosis of interchromosomal insertion of Y chromosome heterochromatin in a family

Bom Yi Lee,Ju Yeon Park,Yeon Woo Lee,Ah Rum Oh,Shin Young Lee,So Yeon Park,Hyun Mee Ryu,Si Won Lee 대한의학유전학회 2017 대한의학유전학회지 Vol.14 No.2

Interchromosomal insertion of Y chromosome heterochromatin in an autosome was identified in a fetus and a family. A fetal karyotype was analyzed as 46,XX,dup(7)(?q22q21.1) in a referred amniocentesis at 16 weeks of gestation for advanced maternal age. In the familial karyotype analyses for identification of der(7), the mother, the first daughter and the maternal grandmother showed the same der(7) as the fetus’s. CBG-banding was positive at 7q22 region of der(7) that indicated inserted material was originated from heterochromatin. The origin of heterochromatic insertion region in der(7) of the fetus and the mother was found in Yq12 region by fluorescent in situ hybridization with a DYZ1 probe. In the specific analysis of Y chromosomal heterochromatic region of ins(7;Y) of the mother, 15 sequence tagged sites from Yp11.3 region including SRY to Yq11.223 region was not detected. Final karyotypes of the mother, the first daughter and the maternal grandmother were reported as 46,XX,der(7)ins(7;Y)(q21.3;q12q12). All female carriers of ins(7;Y) in the family showed normal phenotype and the mother and the maternal grandmother were fertile. A healthy girl was born at term. We report a rare case of familial interchromosomal insertion of Y chromosome heterochromatin detected only in female family members with normal phenotype that was diagnosed prenatally.

Three cases of rare SRY-negative 46,XX testicular disorder of sexual development with complete masculinization and a review of the literature

Lee, Bom Yi,Lee, Shin Young,Lee, Yeon Woo,Kim, Shin Young,Kim, Jin Woo,Ryu, Hyun Mee,Lee, Joong Shik,Park, So Yeon,Seo, Ju Tae Korean Society of Medical Genetics and Genomics 2016 대한의학유전학회지 Vol.13 No.2

Purpose: To identify the clinical characteristics of SRY-negative male patients and genes related to male sex reversal, we performed a retrospective study using cases of 46,XX testicular disorders of sex development with a review of the literature. Materials and Methods:SRY-negative cases of 46,XX testicular disorders of sex development referred for cytogenetic analysis from 1983 to 2013 were examined using clinical findings, seminal analyses, basal hormone profiles, conventional cytogenetic analysis and polymerase chain reaction. Results: Chromosome analysis of cultured peripheral blood cells of 8,386 individuals found 19 cases (0.23%) with 46,XX testicular disorders of sex development. The SRY gene was confirmed to be absent in three of these 19 cases (15.8%). Conclusion: We report three rare cases of SRY-negative 46,XX testicular disorders of sex development. Genes on autosomes and the X chromosome that may have a role in sex determination were deduced through a literature review. These genes, through differences in gene dosage variation, may have a role in sex reversal in the absence of SRY.

An unusual de novo duplication 10p/deletion 10q syndrome: The first case in Korea

Lee, Bom-Yi,Park, Ju-Yeon,Lee, Yeon-Woo,Oh, Ah-Rum,Lee, Shin-Young,Choi, Eun-Young,Kim, Moon-Young,Ryu, Hyun-Mee,Park, So-Yeon Korean Society of Medical Genetics and Genomics 2015 대한의학유전학회지 Vol.12 No.1

We herein report an analysis of a female baby with a de novo dup(10p)/del(10q) chromosomal aberration. A prenatal cytogenetic analysis was performed owing to abnormal ultrasound findings including a choroid plexus cyst, prominent cisterna magna, and a slightly medially displaced stomach. The fetal karyotype showed additional material attached to the terminal region of chromosome 10q. Parental karyotypes were both normal. At birth, the baby showed hypotonia, upslanting palpebral fissures, a nodular back mass, respiratory distress, neonatal jaundice and a suspicious polycystic kidney. We ascertained that the karyotype of the baby was 46,XX,der(10)($pter{\rightarrow}q26.3::p11.2{\rightarrow}pter$) by cytogenetic and molecular cytogenetic analyses including high resolution GTG-and RBG-banding, fluorescence in situ hybridization, comparative genomic hybridization, and short tandem repeat marker analyses. While almost all reported cases of 10p duplication originated from one of the parents with a pericentric inversion, our case is extraordinarily rare as the de novo dup(10p)/del(10q) presumably originated from a rearrangement at the premeiotic stage of the parental germ cell or from parental germline mosaicism.