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.

Paracentric Inversions Found in Prenatal Diagnosis

Lee, Shin Yeong,Lee, Bom Yi,Park, Ju Yeon,Choi, Eun Young,Lee, Yeon Woo,Oh, Ah Rum,Ryu, Hyun Mee,Park, So Yeon Korean Society of Medical Genetics and Genomics 2013 대한의학유전학회지 Vol.10 No.2

Purpose: This study was designed to confirm whether the paracentric inversions of fetuses and parents may be harmless. Materials and methods: We report 10 cases (0.14%) with paracentric inversions among 7,181 prenatal cases observed during prenatal diagnosis performed at Cheil General Hospital between January 2009 and June 2013. We used cytogenetic GTL- and RBG-banding techniques. Results: Of the 10 cases, nine cases were transmitted from each of the parents, and one case was de novo. Nine cases were phenotypically normal up to one month of age after birth. One case was lost to follow-up. We present prenatal diagnosis and follow-up examination of the fetuses with paracentric inversion. Conclusion: Based on our cases, most paracentric inversions are considered to be harmless. The precise identification of paracentric inversions might be clinically important and helpful for genetic counseling.

Smart AAO Membrane for Multiple Gating System

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

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.

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

Lee, Bom-Yi,Park, Ju-Yeon,Lee, Yeon-Woo,Oh, Ah-Rum,Lee, Shin-Young,Park, So-Yeon,Ryu, Hyun-Mee,Lee, Si-Won Korean Society of Medical Genetics and Genomics 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.

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.

Prenatal Diagnosis of the 22q11.2 Duplication Syndrome

Lee, Moon-Hee,Park, So-Yeon,Lee, Bom-Yi,Choi, Eun-Young,Kim, Jin-Woo,Park, Ju-Yeon,Lee, Yeon-Woo,Oh, Ah-Rum,Lee, Shin-Young,Yang, Jae-Hyug,Ryu, Hyun-Mee Korean Society of Medical Genetics 2009 대한의학유전학회지 Vol.6 No.2

22q11.2미세중복 증후군은 학습장애, 선천적 기형에서부터 정상에 이르기까지 다양한 표현형을 나타내는 증후군으로써, 22q11.2 미세결실 증후군인 DiGeorge 증후군과 동일한 위치에서 발생하는 질환이며, 이러한 원인은 유전적 불안정성이 높은 low-copy repeats (LCR) 부위에서 일어나는 유전체의 결손이나 중복에 의해 형성되는 것으로 보고되고 있다. 최근 array CGH가 임상분야에 적용됨에 따라 22q11.2 미세중복 증후군의 진단이 증가되고 있다. 이론적으로 22q11.2 부위의 미세중복이나 미세결실의 빈도는 동일하게 발생해야 하지만, 현재까지 미세결실에 비해 미세중복의 증례보고는 상대적으로 드물며 이는 증상이 없는 경우가 많기 때문인 것으로 알려져 있다. 특히 이전 보고에서 산전에 발견된 미세중복의 증례는 단1례 만이 보고된 바 있다. 저자들은 산전에 진단된 22q11.2 미세중복 증후군 1례의 보고를 통해 유전상담의 중요성과 array CGH의 임상 적용에 관하여 논하고자 한다. The 22q11.2 duplication syndrome is an extremely variable disorder with a phenotype ranging from normal to congenital defects and learning disabilities. Recently, the detection rate of 22q11.2 duplication has been increased by molecular techniques, such as array CGH. In this study, we report a familial case of 22q11.2 duplication detected prenatally. Her first pregnancy was terminated because of 22q11.2 duplication detected incidentally by BAC array CGH. The case was referred due to second pregnancy with same 22q11.2 duplication. We perfomed repeat amniocentesis for karyotype and FISH analysis. Karyotype analysis from amniocytes and parental lymphocytes were normal, while FISH analysis of interphase cells presented a duplication of 22q11.2 in the fetus and phenotypically normal mother. The fetal ultrasound showed grossly normal finding. After genetic counseling about variable phenotype with intrafamilial variability with 50% recurrence rate, the couple decided to continue the pregnancy. The newborn had no apparent congenital abnormalities until 2 weeks after birth. We recommend that family members of patients with a 22q11.2 duplication be tested by the interphase FISH analysis. Also, we point out the importance of genetic counseling and an evaluation of the clinical relevance of diagnostic test results.

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.

Bioinspired Dual Stimuli-Responsive Membranous System with Multiple On–Off Gates

Lee, Bom-yi,Hyun, Seung,Jeon, Gumhye,Kim, Eun Young,Kim, Jinhwan,Kim, Won Jong,Kim, Jin Kon American Chemical Society 2016 ACS APPLIED MATERIALS & INTERFACES Vol.8 No.18

<P>Stimuli-responsive polymers have been widely used for controlled release of several biomolecules. In general, a single stimulus among various stimuli, for instance, temperature, pH, or light, has been used for these polymers. Although some stimuli are applied together, one cannot control each stimulus independently at a given stimulus-responsive polymer. However, to mimic biological system like cell membrane, multiple on off gates utilizing independent control of dual (or multiple) stimuli should be used. Here, we introduce a stimuli-responsive membrane controlled by two orthogonal stimuli. For this purpose, the top and the bottom parts of anodized aluminum oxide membrane walls are independently grafted by thermoresponsive poly(N-isopropylacrylamide) and pH-responsive poly(acrylic acid), respectively, by using surface-initiated atom transfer radical polymerization. The membrane clearly showed two independent on off gates depending on temperature and pH. Furthermore, through light irradiation of two different wavelengths (near-infrared and ultraviolet), temperature and pH were also controlled independently and promptly. Thus, this membrane shows two independent on-off gating of the transport of a model biomolecule of fluorescein isothiocyanate-labeled bovine serum albumin. This strategy suggests the potential of independently modified membrane in layers as stimuli-responsive on off gates for the application of artificial cell membrane.</P>

Dual functional membrane capable of both visual sensing and blocking of waterborne virus

Lee, Bom-yi,Kim, Jinhwan,Kim, Won Jong,Kim, Jin Kon Elsevier 2018 Journal of membrane science Vol.549 No.-

<P><B>Abstract</B></P> <P>Norovirus, which is transmitted by contact with a contaminated object or person who has a norovirus infection, eating contaminated food, or drinking contaminated water, is a highly contagious virus for humans and animals. Among many infection routes, water supply systems from reservoirs or dams become very important due to the possibility of mass infection. Because only a small amount of norovirus (10–100 particles forming unit) can cause the infection, a rapid and easy detection of the virus is required. Once norovirus is detected, the water supply should be quickly shut down.</P> <P>Here, we introduce a dual functional membrane capable of both easy detecting with the naked eye and blocking noroviruses. We employed an anodized aluminum oxide (AAO) membrane with uniform pores. The top and bottom parts of the membrane were grafted with polydiacetylene (PDA) and poly(N-isopropylacrylamide) (PNIPAM), respectively. The top part was further bioconjugated by antibody for feline calicivirus (FCV), which is a model virus whose structure is similar to norovirus. The membrane showed color change from blue to red when the FCVs were attached to the antibodies. Water flux was controlled (turned on and off) by changing water temperature by using the well-known thermosensitive property of PNIPAM chains. On and off control of water flow was quickly established (less than 5s) when a laser with near infrared (NIR) wavelength was turned on and off, respectively. In addition, the membrane completely blocked the virus influx into the water system by antibody binding before visual sensing followed by flow control.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We fabricated a dual functional membrane for waterborne viruses. </LI> <LI> The top and bottom of the membrane were grafted with two functional polymers. </LI> <LI> The membrane showed a rapid color change in the presence of virus. </LI> <LI> The membrane blocked virus influx to water system by antibody binding. </LI> </UL> </P>