Dicistronic GABAB 수용체 발현 벡터의 개발

강해묵 청주대학교 2010 産業科學硏究 Vol.27 No.2

To study the function of heterodimeric GABAB receptor, it is needed to construct the dicistronic vector for the simultaneous expression of GABAB1(GB1) and GABAB2(GB2) subtype genes. To do this, the internal ribosome entry sequence was used and succeed the construction of two types of dicistronic vector of GABAB receptor with the living fluorescent color tag, pGA21 and pGA23. Its expression was confirmed in COS-1 cells. This vectors will be useful to elucidate the physiological role of GABAB receptors in central nervous system.

Cloning and Circadian Expression of Rat Cry1

강해묵,Kyungbae Park 한국분자세포생물학회 2004 Molecules and cells Vol.18 No.2

In mammals, two types of cryptochrome are involved in the regulation of the circadian rhythm. We previously characterized rat Cry2 and its expression in brain tissue [Eun et al. (2001)]. We report here the cloning of another cryptochrome gene, Cry1, from rat brain by reverse-transcription coupled to polymerase chain reaction (RT-PCR), together with rapid-amplification of cDNA ends (RACE). The cloned Cry1 cDNA consists of 2557 nucleotides and has a single open-reading frame encoding a protein of 588 amino acids with start and stop codons. The deduced amino acid sequence was 70% identical with that of rat Cry2. It also showed 95% identity with mouse and human Cry1 but relatively low identity of 82% with that of zebrafish. Circadian expression of rat Cry1 and Cry2 was examined in the suprachiasma nucleus (SCN) and eye by real-time PCR. Expression of Cry1 and Cry2 mRNA in the SCN displayed a circadian rhythm with a peak at the day/night transition, and there was a slightly different circadian pattern of expression of Cry1 and Cry2 in the eye.

형광단백질이 첨부된 GABAB 수용체의 제조 및 발현

강해묵 청주대학교 2007 産業科學硏究 Vol.25 No.1

GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitters in the brain, and require 2 distinct subunits for functioning. This atypical receptor structure is a barrier for investigating the regulation of receptor assembly and trafficking, and elucidating the action mechanism of neuronal modulation in the neuron. It needs to develop a proper GABAB receptor expression vector for persuade the heterogenous dimerlization in the inside of neurons. Thus, the living fluorescent protein, super-ecliptic pHluorin (SEP) tagged GABAB receptor cDNA expression vector was constructed and showed it's successful expression in the COS cells.

Cloning and Expression of Cryptochrome2 in the Bullfrog (Rana catesbeiana)

강해묵,은복기 한국분자세포생물학회 2003 Molecules and cells Vol.16 No.2

형질전환생쥐의 제조 수단으로서 유전자 적중법 및 함정법의 개발 현황

강해묵 한국발생생물학회 2010 발생과 생식 Vol.14 No.4

The construction of transgenic mouse using embryonic stem (ES) cells has been crucial in the functional studies of gene on mouse genome. Gene knockout mice have been powerful for elucidating the function of genes as well as a research model for human diseases. Gene targeting and gene trapping mathods have been the representative technologies for making the knockout mice by using ES cells. Since the gene targeting and the gene trapping methods were independently developed about 20 years ago, it's efficiency and productivity has been improved with a advance of molecular biology. Conventional gene targeting method has been changes to high throughput conditional gene targeting. The combination of the advantage of gene targeting and gene tapping elements allows to extend a spectrum of gene trapping and to improve the efficiency of gene targeting. These advance should be able to produce the mutant with various phenotype to target a certain gene, and in postgenome era they have served as crucial research tools in understanding the functional study of whole genome in mouse.

북방산 개구리 난소의 Cytochrome P450c17유전자 특성

강해묵 한국발생생물학회 2006 발생과 생식 Vol.10 No.2

스테로이드 합성효소 중 17-hydroxylase/17,20-lyase (P450c17)는 progesterone을 17-hydroxyprogesterone을 거쳐 androstenedione으로 변환을 담당하는 효소이다. 양서류 난소에서 스테로이드 합성의 분자적 조절과정의 연구에 사용할 목적으로 북방산 개구리(Rana dybowskii) 난소에서 P450c17 cDNA를 클로닝 하였다. 북방산 개구리 난포세포의 cDNA library 검색을 통해 분리된 약 2.5 kb의 cDNA는 529개의 아미노산을 가진 단일 번역틀을 가지고 있었다. 개구리 P450c17의 아미노산 서열은 Xenopus와는 76%, 닭과는 63%, 그리고 사람과는 약 45%의 동일성을 보여 주였고, 동시에 진화적으로 척추동물에서 매우 잘 보존된 아미노산 서열을 가지고 있었다. 노던 분석에서 개구리의 P450c17 전사체는 난소에서만 2.5 kb와 3.6 kb 크기의 두 종류가 발견되었다. 그리고 개구리 Rana P450c17 cDNA는 비스테로이드 합성 세포인 COS-1세포에서 분명한 17-hydroxylase/17,20-lyase 활성을 주었다. 따라서 클로닝된 개구리 P450c17 유전자는 양서류의 난소에서 스테로이드 합성의 분자적 기작을 연구하는데 매우 유용할 것으로 사료된다.

Circadian Expression of Clock Genes in the Rat Eye and Brain

강해묵,Kyungbae Park 한국분자세포생물학회 2006 Molecules and cells Vol.22 No.3

Characterization of Ovarian Cytochrome (17 /17,20-lyase) in Rana dybowski

강해묵 한국발생생물학회 2006 발생과 생식 Vol.10 No.2

스테로이드 합성효소 중 는 progesterone을 을 거쳐 androstenedione으로 변환을 담당하는 효소이다. 양서류 난소에서 스테로이드 합성의 분자적 조절과정의 연구에 사용할 목적으로 북방산 개구리(Rana dybowskii) 난소에서 cDNA를 클로닝 하였다. 북방산 개구리 난포세포의 cDNA library 검색을 통해 분리된 약 2.5kb의 cDNA는 529개의 아미노산을 가진 단일 번역틀을 가지고 있었다. 개구리 의 아미노산 서열은 Xe [ ] is the key enzyme mediating the conversion of progesterone to , ultimately to androstenedione during steroidogenesis. R. dybowskii's ovarian cDNA was cloned to understand the regulatory mechanism of ovarian steroidogenic pathway at the molecular level in amphibian. A 2.5kb cDNA clone encoding a single open-reading frame with a 519 deduced amino acid was isolated with the screening of ovarian cDNA library. This sequence contained the three highly conserved domains as seen in of other species. The comparison of amino acid sequence of Rana with other animal's showed relatively high identity with 76% in Xenopus, 63% in chicken, 60% in rainbow trout, and 45% in human. Phylogenic analysis also indicated that Rana gene was evolutionary well conserved among vertebrate. Northern analysis indicated that the two different sizes of transcripts with approximately 2.5 and 3.6kb were detected in ovary tissue, but not in other tissues. The expression vector of Rana clearly showed the activity converting the exogenous progesterone into in the nonsteroidogenic COS-1 cells. Therefore, Rana cDNA is very useful to investigate the molecular mechanism of the ovarian steroidogenesis in amphibian.

Dibutyryl Cyclic AMP로 처리된 생쥐난자의 수정능에 관한 연구

강해묵,이영기,조완규 한국통합생물학회 1988 동물학회지 Vol.31 No.1

The present study was carried out to examine the fertilizability of the mouse oocytes pre-ex-posed to dbcAMP which is a well-known inhibitor of the oocyte maturation. The oocytes once cultured in the dbcMP-containing medium for a certain length of, time were cultivated in the dbcMp-free medium to induced the maturation, then mixed with sperms, and observed following culture for 24 hours. The fertilization rate of cocytes was judged by the index of the number of 2-cell embryo developed 24hr following insemination. The fertilization rate of the oocyte previously incubated with dbcAMP (100 g/ml) for 2, 4, 8 16 hours was 32.3, 14.5, 4.7 and 8.8%, respectively, while that of the control was 53.3% indicating that the fertilizability was decreased as a function of time exposed to dbcAMP. The pretreatment of dbcMP, however, didn't affect the process of sperm penetration to egg. In addition, there is no prominent changes in the morphological architecture of fertielized eggs which has been exposed to dbcAMP as revealed by electron microscopic observation. Consequendy, it can be concluded that the mouse cocytes once inhibited their maturation by dbcMP may retain, in some extent, the fertilizability, although most of the fertilized egg may not proceed to further development because of the failure of pronucleus formation. dbcAMP에 의해 성숙이 억제된 생쥐난자의 수정능을 조사하기 위해 본 실험을 행하였다. dbcAMP로 일시 성숙이 억제되었던 난자를 배양액내에서 정자와 섞고 24시간 배양한 후 발생한 2세포기의 배아형성, 정자의 관입, 전액형성을 조사하여 2세포기로 배아발생이 진척된 것을 수정율의 기준으로 정하였다. dbcAMP를 처리하지 않은 난자는 약 53.3%의 수정율을 보였으며,dbcAMP가 함유되 배양액에서 배양한 후 기본 배양액에서 성숙시킨 난자들의 수정율은 dbcAMP의 처리시간에 비례하여 낮으나, 정자의 관입은 정상적으로 일어났다. 전자현미경적 관찰에 의하면 dbcAMP 처리는 난자의 미세구조에 어떠한 변화도 야기하지 않았다. 따라서 dbcAMP를 사전처리하여 성숙을 억제한 난자라할지라도 어느 정도의 수정능력을 보유하고 있다고 사료된다.

Characterization of Ovarian Cytochrome $P450_{C17}$ (17 ${\alpha}-hydroxylase$/17,20-lyase) in Rana dybowski

강해묵,Kang, Hae-Mook The Korean Society of Developmental Biology 2006 발생과 생식 Vol.10 No.2

스테로이드 합성효소 중 $17\;{\alpha}-hydroxylase/17,20-lyase(P450_{C17})$는 progesterone을 $17\;{\alpha}-hydroxyprogesterone$을 거쳐 androstenedione으로 변환을 담당하는 효소이다. 양서류 난소에서 스테로이드 합성의 분자적 조절과정의 연구에 사용할 목적으로 북방산 개구리(Rana dybowskii) 난소에서 $P450_{C17}$ cDNA를 클로닝 하였다. 북방산 개구리 난포세포의 cDNA library 검색을 통해 분리된 약 2.5kb의 cDNA는 529개의 아미노산을 가진 단일 번역틀을 가지고 있었다. 개구리 $P450_{C17}$의 아미노산 서열은 Xenopus와는 76%, 닭과는 63%, 그리고 사람과는 약 45%의 동일성을 보여 주였고, 동시에 진화적으로 척추동물에서 매우 잘 보존된 아미노산 서열을 가지고 있었다. 노던 분석에서 개구리의 $P450_{C17}$ 전사체는 난소에서만 2.5kb와 3.6kb 크기의 두 종류가 발견되었다. 그리고 개구리 Rana $P450_{C17}$ cDNA는 비스테로이드 합성 세포인 COS-1세포에서 분명한 $17\;{\alpha}-hydroxylase/17,20-lyase$ 활성을 주었다. 따라서 클로닝된 개구리 $P450_{C17}$ 유전자는 양서류의 난소에서 스테로이드 합성의 분자적 기작을 연구하는데 매우 유용할 것으로 사료된다. [ $17\;{\alpha}-hydroxylase/17,20-lyase(P450_{C17})$ ] is the key enzyme mediating the conversion of progesterone to $17\;{\alpha}-hydroxyprogesterone$, ultimately to androstenedione during steroidogenesis. R. dybowskii's ovarian $P450_{C17}$ cDNA was cloned to understand the regulatory mechanism of ovarian steroidogenic pathway at the molecular level in amphibian. A 2.5kb cDNA clone encoding a single open-reading frame with a 519 deduced amino acid was isolated with the screening of ovarian cDNA library. This sequence contained the three highly conserved domains as seen in $P450_{C17}$ of other species. The comparison of amino acid sequence of Rana $P450_{C17}$ with other animal's $P450_{C17}$ showed relatively high identity with 76% in Xenopus, 63% in chicken, 60% in rainbow trout, and 45% in human. Phylogenic analysis also indicated that Rana $P450_{C17}$ gene was evolutionary well conserved among vertebrate. Northern analysis indicated that the two different sizes of $P450_{C17}$ transcripts with approximately 2.5 and 3.6kb were detected in ovary tissue, but not in other tissues. The expression vector of Rana $P450_{C17}$ clearly showed the $17\;{\alpha}-hydroxylase$ activity converting the exogenous progesterone into $17\;{\alpha}-hydroxyprogesterone$ in the nonsteroidogenic COS-1 cells. Therefore, Rana $P450_{C17}$ cDNA is very useful to investigate the molecular mechanism of the ovarian steroidogenesis in amphibian.