지적 장애 관련 cnu071 유전자의 분리 및 기능 분석

이솔비 忠南大學校 大學院 2016 국내석사

Intellectual disability (ID), also called mental retardation (MR), is a generalized neurodevelopmental disorder defined by an IQ score under 70. It is characterized by deficited learning and adaptive behaviors. ID is caused by genetic factors; chromosomal abnormalities, genes defects, and environmental problems, such as social problems and infections. However, the exact causes and treatments for the ID are yet to be well defined. Here I have studied a gene named as cnu071, which was identified as a candidate gene for ID, from human patients using the Genome Wide Association Studies (GWAS). During sequence analysis, I found that there was 90% homology between the human and zebrafish cnu071 proteins. Using the whole-mount in situ hybridization (WISH) method, I examined the mRNA expression pattern of the cnu071 during zebrafish development. I also established cnu071 knockout (KO) zebrafish using CRISPR/Cas9 system. I found a phenotype of swim bladder defect in cnu071 KO mutant zebrafish. Some of fish were able to survive up to the adult stage at low frequency. To understand the molecular mechanism, I analyzed expression of several molecular markers in the cnu071 KO mutant, using WISH method. There were no significant difference in the expressions of these molecular markers at early larval stages. However, at the adult stage, I found a significant change in the size of brain in cnu071 KO fish, compared to that of wild type fish. In relation to ID patients, I tried to test neurobehavioral changes in the cnu071 KO fish. In social behavior test, cnu071 KO fish showed a significant reduction in their social interaction. From these results, I suggest that cnu071 is a causative gene for ID in humans.

제브라피쉬 배발생을 이용한 인간유전체 대량 기능 연구

김동일 충남대학교 대학원 2007 국내석사

In the post-genome era, researches about gene function have been actively progressing and new model system is urgently required for high throughput functional screen at a genome level. Thus I challenged a genome-wide screen against the genome-wide human UniGene Collections. Currently, more than 10,000 human full-length cDNAs are available from the Korean UniGene Information (KUGI). After in silico screen of novel genes through database search, I performed overexpression experiments by microinjecting the single individual human genes into the zebrafish embryos. So far, I tried more than 1,000 human full-length cDNAs and isolated several novel functional genes at the level of 2% hit. Zebrafish embryos displayed variable phenotypes from developmental defects to neuronal cell death after microinjection. Most of these genes were involved in not only developmental process but also diseases, such as cancer. After these phenotype-based screening, homologous zebrafish genes were also cloned and further functional studies were performed in detail by overexpression, expression pattern analysis, and antisense KO approaches. In conclusion, I suggest that this phenotype-based large-scale functional screening of human genes using zebrafish system is a very powerful tool to find novel functional genes in post-genome era.

제브라피쉬 초기발생에 있어서 신경세포와 혈관세포 간의 상호작용에 대한 연구

함형걸 충남대학교 대학원 2007 국내석사

Blood vessels and nerves are structurally similar complex branched systems. Their guidance must be exquisitely regulated to ensure proper wiring of both networks. Recent results showed that specialized endothelial cells, resembling axonal growth cones, form the tips of growing capillaries. These endothelial tip cells guide outgrowing capillaries in response to gradients of extra cellular matrix-bound vascular endothelial growth factor. Several axon guidance molecules, including Semaphorins, Netrins, Ephrins and Slits, have also been implicated in vessel path finding and network formation. My current study focuses on the possible interaction between primary motoneurons and intersomitic vessels during early development. I established a double-transgenic zebrafish with GFP expression in blood vessel cells and RFP expression in motoneurons, enabling me to examine their development at a cellular level in live animals. With this powerful experimental animal system, I tested the involvement of midline signaling in motoneuron and vessel development. Cyclopamine, a chemical inhibitor of the Shh pathway, inhibited both motoneuron and vessel development. Specific inhibition of motoneuron development with antisense olig2 morpholino knockdown affected intersegmental vessel formation. To identify signaling guidance molecule, I analyzed expression of several candidate genes and found that slit-3 gene is down-regulated in motoneurons. These findings suggest that motoneuron has a possible interaction with vessel cells during early embryo development.

화학 유전체 접근법에 의한 세포에서의 H-Ras와 PRL-3 표적 유전자의 규명

이수경 충남대학교 대학원 2007 국내박사

화학 유전체 접근법은 넓은 범위의 생물학적 과정의 원리를 설명하기 위해서 발달되었다. 전통적은 유전학에서의 특정 유전자 돌연변이 유도를 대신하여, 화학 유전체학에서는, 단백질의 기능을 화학 리간드를 이용하여 억제시키거나 활성화 시킨다. 그들은 빠르게 세포 안으로 투과되어, 표적 단백질과 상호작용하여 기능 손실 혹은 기능 획득 표현형을 창출할 수 있다. 이 논문에서, 발암 유전자인 H-Ras와 PRL-3에 의해 조절되는 유전자를 규명하고자 하였다. H-Ras와 PRL-3 단백질은 활성화를 위해 파네실레이션 과정을 거친다. 그래서 파네실트랜스퍼라아제의 화학적 억제제는 세포에서 H-Ras와 PRL-3의 기능을 규명하는데 매우 유용하다. H-Ras는 암 성장유지와 신생혈관 형성에 중요하다. 파네실트랜스퍼라아제에 의한 H-Ras의 파네실레이션이 H-Ras 기능을 위해 매우 중요한 과정이기 때문에 파네실트랜스퍼라아제 억제제는 H-Ras의 기능에 영향을 미칠 수 있다. 파네실트랜스퍼라아제 억제제의 탐색 결과, 아데미노라이드알디(AP-Rd)가 억제제로 규명되었다. (IC_(50) 1 uM). SCH66336과 LB42908은 포지티브 대조군으로 사용되었으며 이 화합물은 각각 Schering Plough사와 LG chemical사에서 개발되었다. 사용된 모든 파네실트랜스퍼라아제 억제제는 H-Ras의 파네실레이션과 EPK 활성을 억제시킬 수 있었다. H-Ras 하위 단계의 유도분자를 규명하기 위해서, mock 혹은 H-Ras가 형질 전환된 Rat2 세포의 유전자 발현 양상을 파네실트랜스퍼라아제 억제제의 처리 혹은 무처리군을 가지고 분석하였다. 분석을 통하여 H-Ras 의존적으로 증가 혹은 감소하는 유전자를 규명하였다. 이 결과를 기초로 본 연구는 H-Ras와 MMPs발현의 관계를 RT-PCR, western blot 그리고 zymographic 분석을 통하여 확인하고자 하였다. MMP-9과 MMO-13의 발현과 활성은 H-Ras가 형질전환된 세포에서 확연하게 증가되었고 파네실트랜스퍼라아제 억제제의 처리에 의해서 그들의 활성이 억제되었다. 더나아가, 파네실트랜스퍼라아제 억제제 혹은 MMP-9/13 억제제에 의해서 신생혈관이 억제된다는 것을 인간 혈관내피세포(HUVECs)의 튜브형성과 동물모델로써 제브라피쉬를 이용하여 확인하였다. 이러한 결과들은 H-Ras에 의한 MMO-9뿐만 아니라 MMP-13의 활성이 신생혈관형성에 관여한다는 것을 지지해준다. PRL-3(Phosphatase of regenerating liver-3)는 단백질 타이로신 포스퍼티아제(PTP)이며 그들의 활성을 위해서는 H-Ras와 유사하게 파네실레이션에 의해 변이된다. 부가적으로 PRL-3 단백질은 암세포의 이동과 전이에 깊이 관여하고 있다. 인간 대장암 유래 세포(DLD-1)를 사용하여, PRL-3의 발현이 암세포의 이동과 전이를 촉진시킨다는 것을 증명하였다. 파네실트랜스터라아제 억제제를 포함한 PRL-e의 특정 억제제들은 세포 이동과 침윤을 억제시킬 수 있다. PRL-3의 하위단계 표적인자를 규명하기 위해서, mock과 PRL-3가 형질전환된 세포간의 유전자 발현양상을 cDNA microarray를 이용하여 분석하였다. 그 분석으로부터 MMP-7, integrin alpha E, tubulin beta, 그리고 tetraspanins와 같은 세포 부착과 운동성에 관련된 많은 유전자들이 PRL-3에 의해 조절된다는 것을 알게 되었다. 종합적으로, 본 연구는 화학유전체 접근법이 여러 생물학적 과정에 대한 기작을 설명하기에 매우 유용하다는 것을 시사해 주고 있다. Chemical genetic approach has been developed to elucidate the principles of a wide range of biological processes. In chemical genetics, instead of site-specific mutation in traditional genetics, function of protein is inhibited or activated by using small chemicals. They can rapidly penetrate into cells, interact with their target proteins, and create loss-of-function (or gain-of-function) phenotypes. In this study, it has been tried to identify and characterize the genes that are regulated by oncogenic H-Ras and PRL-3. Both H-Ras and PRL-3 are farnesylated for their activation. Therefore, chemical inhibitor of farnesyl transferase (FPTase) will be very useful to understand function of H-Ras and PRL-3 in cells. H-Ras has essential roles for tumor maintenance and angiogenesis. Because farnesylation of the H-Ras by FPTase is acritical step for the H-Ras function, FPTase inhibitor (FPTI) could affect H-Ras functions. From the screening of FPTI, arteminolide Rd (AP-Rd) was identified as a potent inhibitor (IC50 is 1 uM). SCH66336 and LB42908 are used as positive controls which are developed by Schering Plough Company and LG chemicals Company, respectively. All the used FPTI could block H-Ras farnesylation and ERK activation. To identify downstream effectors of H-Ras, it has been carried out to analyze the gene expression profile between mock and H-RasV12 transfected Rat2 cells in the absence or presence of FPTIs. From this, I identified H-Ras-dependent up- or down-regulated genes. On the basis of the results, this study was focused to solve the relationship between H-Ras and MMPs expression using RT-PCR, western bolt and zymographic analysis. Expressions and activations of MMP-9 and MMP-13 were significantly enhanced in H-Ras transformed cells and their activities were abolished by treatment with FPTIs. Furthermore, it was confirmed that angiogenesis could be blocked by inhibitor of MMP-9 /13 or FPTIs using capillary formation assay with HUVECs or using zebrafish as a model animal. These results support that activation of MMP-13 as well as MMP-9 induced by H-Ras is involved in angiogenesis. Phosphatase of regenerating liver-3 (PRL-3) is a protein tyrosine phosphatase (PTP) and modified by farnesylation for its activity like H-Ras. In addition, it has been strongly implicated in tumor cell migration and metastasis. Using a human colon cancerderived cell system (DLD-1 cells), this study proved that the overexpression of PRL-3 could promote tumor cell migration and invasion. The specific inhibitors of PRL-3 including FPTI could block tumor cell migration and invasion. To identify downstream effectors of PRL-3, it has carried out to analyze the gene expression profile between mock and PRL-3 transfected cells by using DNA microarray. From this analysis it was found that many of genes encoding cell adhesion and motility such as MMP-7, integrin alpha E, tubulin beta, and tetraspanins were regulated by PRL-3. Overall, this study supports that chemical genetic approach is very useful to elucidate the mechanisms of several biological processes.

Regulatory factors of mophological differentiation in streptomyces griseus

이기은 충남대학교 대학원 2007 국내석사

To understand the morphological events during sporulation of Streptomyces griseus, I characterized a nonsporulating mutant, SKK1003 by genetic complementation. The mutant has a defect in accumulating the eshA transcript and protein during sporulation. The mutant was restored to sporulate by several pieces of the genomic DNA from the wild-type strain in low copy number vector, pXE4. The genetic mapping and sequence determination of the gene fragments revealed that the mutant contained a point mutation in the open reading frame of the adpA gene, which serves as a key transcription regulator during aerial mycelium formation and streptomycin production. To test whether eshA is regulated by adpA, I carried out the gel mobility shift assay using the recombinant AdpA. The AdpA bound to the upstream regulatory region of eshA in a concentration-dependent manner. This result demonstrated that eshA is another member of the adpA regulon.

Zebrafish 혈관발생에서 ldb2 유전자의 발현 및 기능 분석

최동훈 충남대학교 보건·바이오산업기술대학원 2007 국내석사

LIM homeodomain proteins are developmental regulators whose functions depend on synergism with LIM domain binding proteins (Ldb proteins). LIM homeodomain has double zinc-finger motifs which have been shown to mediate protein?protein interactions. Ldb proteins are transcriptional activators that associate with the LIM homeoproteins and coordinate in target gene transcription. LIM homeodomain proteins and Ldb proteins play critical roles in vertebrate development and cellular differentiation. To study the in vivo function of Ldb proteins, I isolated zebrafish ldb2 gene. The zebrafish Ldb2 protein was composed of 385 amino acids and showed a 87 % amino acid identity with human and mouse counterparts. ldb2 is expressed ubiquitously at early gastrulation stage, but its expression is restricted to vessel cells at later stages, implying its possible role in vessel development. Angiogenesis is a process of which new blood vessels develop by sprouting of preexisting vessels. Knock-down of ldb2 gene, using antisense morpholino oligonucleotides, inhibited normal sprouting of intersegmental vessels and showed severe defects in pathfinding of vessel cells. These results suggest that ldb2 gene has a critical role in zebrafish angiogenesis.

ESTs 데이터베이스를 이용한 파킨슨병과 관련된 유전자 발굴

정소영 충남대학교 대학원 2007 국내석사

Licochalcone A에 의한 파골세포 형성 및 골 흡수 활성의 억제효과

김순남 충남대학교 대학원 2008 국내석사

Zebrafish로부터 새로운 chemokine samdori의 분리 및 유전자 발현 분석

최정화 충남대학교 대학원 2008 국내석사

Through an insertional mutagenesis screen, I isolated a novel chemokine gene, samdori. Chemokines are small secreted molecules that have conserved structure. They act as mediators of cellular migration in embryogenesis, and of leukocyte migration in immune surveillance.On the basis of DNA walking method, I mapped the samdori locus on the chromosome 4. From the zebrafish genome database search, I further identified the existence of additional members of the samdori gene family; samdori 1, 2, 3, 4, 5a and 5b. I cloned these individual family members by RT-PCR and 5'-/3'-RACE method and analysed their spatiotemporal expression pattern during development. All of these samdori family genes are specifically expressed in the central nervous system. To identify the cell type of samdori-expressing cells in nervous system, I used the neurogenic mutant, mind bomb, and found that most of these cells are neuronal cells, suggesting their physiological role as a new family of neurokine in nervous system. Interestingly, samdori 2 is specifically expressed in adult habenula which is a central part of brain function.

Establishment of in vivo animal model for the myelination diseases

Jae-Ho Ryu 충남대학교 분석과학기술대학원 2012 국내석사