• Journal of Plant Biotechnology
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• 제31권4호
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• pp.267-271
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• 2004

국내 고구마 율미 품종의 배발생 캘러스를 Agrobacterium 매개 방법을 이용하여 형질전환 식물체를 개발하였다. 배발생 캘러스를 7일 동안 전배양 한 후 Agrobacterium과 2일 간 공동배양할 경우 일시적인 형질전환 효율이 가장 높았다. Agrobacterium과의 공동배양 후 배발생 캘러스를 1mg/L 2,4-D, 100mg/L kanamycin, 400mg/L claforan 이 첨가된 선발배지에서 4주 간격으로 계대배양하였다. 선발된 kanamycin 저항성 캘러스를 2,4-D를 제거한 선발배지로 옮겨 체세포배를 유도하였으며 이후 소식물체로 발달하였다. Southern 분석으로 1-3 copy의 GUS 유전자가 고구마 염색체내로 도입되었음을 확인하였다. 또한 조직학적 분석으로 GUS 유전자가 형질전환 고구마의 배발생 캘러스, 재분화 식물체의 잎, 엽병, 및 뿌리 조직에서 강하게 발현됨을 알 수 있었다.

• Journal of Plant Biotechnology
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• 제30권3호
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• pp.233-239
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• 2003

We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

• 한국양식학회지
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• 제7권1호
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• pp.41-54
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• 1994

E. coli의 \beta-galactosidase$ 유전자를 미꾸라지 수정난에 미세현미 주입하고 이를 분석함으로써 미꾸라지에 외래 유전자 이식을 위한 reporter 유전자로서의 유용성을 검토하였다 X-gal 염객분석, 4-methylumbelliferyl-$\beta$-D-galactoside (MUG) 분석을 수행한 결과 유전자 이식 처리군 및 대조군에서 모두 \beta-galactosidase$의 활성이 관찰되었으며 PCR, dot blot 및 southern blot분석결과 역시 유전자 이식 처리군과 대조군에서 모두 유사한 양상을 나타내었다. 처리군 및 대조군의 PCR product의 염기서열은 E. coli의 \beta-galactosidase$ 유전자와 매우 높은 homology를 갖고 있었으며 pH에 따른 X-gal 염색 분석을 수행한 결과 미꾸라지에 관찰되는 본 효소는 pH 4.5에서 가장 높은 활성을 나타내었다. 따라서 앞으로 미꾸라지를 대상으로 한 외래 유전자 이식시 E. coli의 \beta-galactosidase$ 유전자의 reporter 유전자로서의 사용은 신중한 재검토가 이루어져야만 할 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.707-714
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• 2004

How to improve the cell culture method on scaffolds is important in the tissue engineering fileld. In this study, we optimized seeding and culture methods to vascular smooth muscle cells (VSMCs) on biodegradable polymer scaffold. The primary culture of VSMCs obtained from canine external jugular vein was accomplished by applying the explant-derived method. The primary cultured VSMCs were seeded into scaffolds and then cultured by using various different methods; static or dynamic seeding, static or dynamic culture. The difference in proliferative response of VSMCs was analyzed with an alamar blue assay. Cell-polymer construct was examined by histochemical method and scanning electron microscopy. Mesh type scaffold ($10 \times 10 \times0.4 mm$) was made of polyglycolic acid (PGA) suture thread. The PGA mesh type scaffold was 45% in porosity, and 0.03 g in weight. The primary cultured VSMCs were confirmed with immunohistochemical staining using monoclonal anti-$\alpha$-smooth muscle actin. The density and distribution of proliferated VSMCs within the scaffold and cellular adherence on the surface of the scaffold showed better results in the static seeding condition than in the dynamic condition. Under the same condition of seeding method as the static condition, the dynamic culture condition showed enhanced proliferation rates of the VSMCs when compared to the static culture condition. In conclusion, to improve the VSMCs proliferation in vitro, static seeding is better than the dynamic condition. In the culture condition, however, culture under the dynamic status is better than the static condition. This was a pilot study to manufacture artificial vascular vessel by tissue engineering.

• 식물조직배양학회지
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• 제25권5호
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• pp.329-333
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• 1998

Escherichia coli의 $\beta$-glucuronidase (GUS) 유전자를 고구마의 배발생세포괴에 particle bombardment로 도입하여 재분화 식물체에 발현시켰다. CaMV35S-GUS 융합유전자와 선발표지로서 neomycin phosphotransferase유전자가 들어있는 binary 운반체 pBI121 DNA를 텅스텐 입자로 코팅하여 정단분열 조직 유래의 배발생 세포괴에 bombarding하였다. Bombarding된 세포괴를 1mg/L 2,4-D와 100mg/L kanamycin이 첨가된 MS 배지로 옮겨 한달 간격으로 6개월동안 계대배양하였다. Kanamycin 저항성 캘러스를 0.03mg/L 2iP, 0.03 mg/L ABA 및 50 mg/L kanamycin이 들어있는 MS 배지로 옮겨 체세포배를 유도하였고, kanamycin이 첨가되지 않은 MS 기본배지에서 식물체로 발달시켰다. 토양에서 생육중인 6개체의 식물을 대상으로 PCR과 northern분석을 수행한 결과 GUS 유전자가 식물체 genome에 안정적으로 도입, 발현되었음이 확인되었다. 조직화학적 분석으로 GUS 유전자가 형질전환 식물체에서 발현됨을 밝혔다.

• Applied Biological Chemistry
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• 제39권4호
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• pp.260-264
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• 1996

Cymbidium속 난의 형질전환계를 확립하기 위해서, microprojectile bombardment 방법을 이용하여 춘란(Cymbidium virescence)의 rhizome 조직세포에 외래 유전자를 도입하는 조건을 검토하였다. 각 parameter별 적정조건으로서 텅스텐 입자의 크기는 $1.11\;{\mu}m$, He 가스 압력은 $77.33kg/cm^2$, gap distance는 6.35mm, target distance는 7.0cm 이었다. DNA피복입자를 투사한 $400{\mu}m$ 두께의 rhizome 절편을 2개월간 배양한 뒤 GUS 활성을 조사한 결과 이 유전자가 발현되는 세포들이 관찰되었다. Kanamycin (100 mg/L)을 첨가한 배지에서 6개월 동안 선택배양을 통해 얻은 rhizome의 DNA를 PCR로 분석한 결과 nptII 유전자의 삽입을 확인할 수 있었다.

• 생명과학회지
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• 제14권4호
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• pp.657-663
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• 2004

고추에서 클로닝된 sesquiterpene cyclase (CASC) promoter에 cinnamic acid 4-hydroxylase (C4H)유전자를 클로닝하여 담배에 형질전환하였다. 형질전환체의 분석은 PCR, Southern blot분석으로 하였으며, CASC promoter의 작동여부를 조사하기 위하여 GUS histochemical assay를 하였다. 스트레스에 반응한다고 알려진 CASC promoter를 C4H gene과 fusion하여 C4H activity를 조사하여 스트레스에 반응하는 정도를 조사하였다. 최종적인 형질전환체로 확인된 개체는 평균 16.4% 정도였으며, 각 promoter별로 약간의 편차를 보였다. C4H activity 조사시 스트레스를 주지 않았을 때 대조구가 비슷한 양상을 나타내었다. 스트레스를 주었을 때 대조구와 비교하여 promoter 3번은 1.3배 activity가 높아졌으며 4번은 1.4배, 6은 2배, cyc 600은 1.1배 높아졌다. 이런 결과로 보아 이 promoter들은 UV에 반응하는 promoter이며 앞으로 stress에 저항하는 유전자의 적당한 promoter로, 타작물에 적용하여 stress저항성 작물의 육성 시 매우 유용하게 사용될 것으로 사료된다.

• Journal of Plant Biotechnology
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• 제32권2호
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• pp.91-95
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• 2005

옥수수 미숙배배양과 Agrobacterium tumefaciens공동배양법에 의해 형질전환체를 생산하였다. Hi II계통의 미숙배를 Ubiquitin 1 promoter-GUS유전자와 선발마커로서 nptII 유전자로 제작된 pPTN290벡터를 C58C1에 도입한 후 형질전환 균주로 사용하였다. 7개의 paromomycin저항성 배 발생캘러스를 얻었으며, GUS양성반응을 나타내는 7개의 독립적인 식물체를 얻었다. Southern분석법에 의하여 $T_1$세대 식물체로부터 nptII유전자가 안정적으로 도입되어 있음을 확인하였다.

• Journal of Ginseng Research
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• 제41권3호
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• pp.419-427
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• 2017

Background: Glycosylation of natural compounds increases the diversity of secondary metabolites. Glycosylation steps are implicated not only in plant growth and development, but also in plant defense responses. Although the activities of uridine-dependent glycosyltransferases (UGTs) have long been recognized, and genes encoding them in several higher plants have been identified, the specific functions of UGTs in planta remain largely unknown. Methods: Spatial and temporal patterns of gene expression were analyzed by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and GUS histochemical assay. In planta transformation in heterologous Arabidopsis was generated by floral dipping using Agrobacterium tumefaciens (C58C1). Protein localization was analyzed by confocal microscopy via fluorescent protein tagging. Results: PgUGT72AL1 was highly expressed in the rhizome, upper root, and youngest leaf compared with the other organs. GUS staining of the promoter: GUS fusion revealed high expression in different organs, including axillary leaf branch. Overexpression of PgUGT72AL1 resulted in a fused organ in the axillary leaf branch. Conclusion: PgUGT72AL1, which is phylogenetically close to PgUGT71A27, is involved in the production of ginsenoside compound K. Considering that compound K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of ginsenosides in ginseng plant growth and development. The organ fusion phenotype could be caused by the defective growth of cells in the boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, and requires further analysis.

• 대한한방내과학회지
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• 제37권4호
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• pp.591-600
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• 2016

Objective: This study aimed to examine the relaxation effects and underlying mechanisms of Cynomorii herba (CH) extract in isolated rabbit corpus cavernous tissues.Methods: We experimented with CH extract (0.01-3.0 mg/mL). Nω-nitro-L-arginine (L-NNA) was experimented before the CH extract to contracted strips induced by phenylephrine (PE, 1 μM)and compared with nonexperimented. In addition, we experimented with calcium chloride (Ca²⁺, 1 mM) after pretreatment of the CH extract in Ca²⁺-free Krebs-Ringer solution to contracted strips induced by PE. The cell viability and nitric oxide (NO) concentration of human umbilical vein endothelial cells (HUVECs) were measured by an methylthiazol-2-yl-2, 5-diphenyl tetrazoliumbromide (MTT) assay and Griess reagent system. The ratio of smooth muscles to collagen fibers, in addition to eNOS- and PDE-5-positive reactions, was examined by histochemical and immunohistochemical staining.Results: The CH extract significantly induced the relaxation of the cavernous strips, and the pretreatment with L-NNA inhibited CH extract-induced relaxation. The L-NNA pretreatment reduced the increased contraction induced by the addition of Ca²⁺in Ca²⁺-free solution. Furthermore, the NO concentration of the HUVECs increased. When the CH extract was applied to the corpus cavernosum of the penis (CCP) of Sprague Dawley rats, the ratio of smooth muscles to collagen fibers by PE and the formation of eNOS around the helicine artery increased. However, the CH extract treatment decreased PDE-5 positive reactions.Conclusions: These results show that the relaxation effects induced by the CH extract are associated with the suppression of the influx of extracellular Ca²⁺ via the production of NO and eNOS and inhibition of PDE-5.