• Title/Summary/Keyword: expression vectors

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Expression of Recombinant Human Follicle-stimulating Hormone in the Chinese Hamster Ovary Cell

  • Park, Ji-Hyun;Kim, Nam-Hyung;Hosup Shim;Kim, Teoan
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.100-100
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    • 2002
  • As an preliminary experiment for making transgenic animals producing human follicle stimulating hormone (hFSH), we tried to express recombinant hFSH gene in vitro. hFSH is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. Genes encoding the common gonadotrophin alpha subunit and FSH-specific beta subunit were inserted into retroviral vectors under the control of the rat beta actin promoter. Gene transfer to the Chinese hamster ovary (CHO) cells was done by infection of the retroviruses harvested from PT67 packaging cells transfected with recombinant retrovirus vector DNA. After selection with G4l8, PCR and RT-PCR analyses of the G4l8-resistant CHO cells showed successful transfer and expression of both ${\alpha}$ and ${\beta}$ fragments of the FSH gene.

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Modification of amylose content of sweetpotato starch by RNAi technique

  • Shimada, Takiko;Otani, Motoyasu;Hamada, Tatsurou;Kim, Sun-Hyung
    • Proceedings of the Korean Society of Plant Biotechnology Conference
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    • 2005.11a
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    • pp.351-355
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    • 2005
  • In the storage roots of sweetpotato (Ipomoea batatas (L.) Lam. cv. Kokei 14), 10 to 20% of starch is essentially unbranched linear amylose and the other major component is branched amylopectin. Amylose is produced by the enzyme GBSSI (granule bound starch synthase I), whereas amylopectin is produced by a concerted action of soluble starch synthase and starch branching enzymes (SBEI and SBEII). We constructed double-stranded RNA (dsRNA) interference vectors of GBSSI and IbSBEII and introduced them into sweetpotato genome via Agrobacterium-mediated gene transformation. The endogenous GBSSI expression was inhibited by dsRNA of GBSSI in 73 % of transgenic plants giving rise to the storage tubers containing amylopectin but not amylose. On the other hand, all sweetpotato plants transformed with dsRNA of IbSBEII contained a larger amount of amylose than the non-transgenic control (up to 25% compared to 10% in the controls). The RNA interference (RNAi) is effectively inhibited the gene expression in thestarch metabolic pathway and modified the characteristics of starch in sweetpotato.

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Recognition of Facial Emotion Using Multi-scale LBP (멀티스케일 LBP를 이용한 얼굴 감정 인식)

  • Won, Chulho
    • Journal of Korea Multimedia Society
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    • v.17 no.12
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    • pp.1383-1392
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    • 2014
  • In this paper, we proposed a method to automatically determine the optimal radius through multi-scale LBP operation generalizing the size of radius variation and boosting learning in facial emotion recognition. When we looked at the distribution of features vectors, the most common was $LBP_{8.1}$ of 31% and sum of $LBP_{8.1}$ and $LBP_{8.2}$ was 57.5%, $LBP_{8.3}$, $LBP_{8.4}$, and $LBP_{8.5}$ were respectively 18.5%, 12.0%, and 12.0%. It was found that the patterns of relatively greater radius express characteristics of face well. In case of normal and anger, $LBP_{8.1}$ and $LBP_{8.2}$ were mainly distributed. The distribution of $LBP_{8.3}$ is greater than or equal to the that of $LBP_{8.1}$ in laugh and surprise. It was found that the radius greater than 1 or 2 was useful for a specific emotion recognition. The facial expression recognition rate of proposed multi-scale LBP method was 97.5%. This showed the superiority of proposed method and it was confirmed through various experiments.

Closed-form Expression for the Symbol Error Probability of Orthogonal Space-Time Block Codes with Quadrature Amplitude Modulation (QAM 변조방식을 갖는 직교 시공간 블록 부호의 심볼 오율)

  • 김상효;강익선;노종선
    • The Journal of Korean Institute of Communications and Information Sciences
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    • v.28 no.6C
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    • pp.561-569
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    • 2003
  • In this paper, for my linear orthogonal space-time block including the orthogonal space-time codes introduced by Alamouti[1], Tarokh[14], and Xia[11], the exact expression for the pairwise error probability in the slow Rayleigh fading channel is derived in terms of the message symbol distance between two message vectors rather than the codeword symbol distance between two transmitted codeword matrices. Using the one-dimensional component symbol error probability, the exact closed form expressions for the symbol error probability of linear orthogonal space-time codes are derived for QPSK, 16-QAM, 64-QAM, and 256-QAM.

Overexpression of ER Resident Molecular Chaperones and Characterization of Their Interaction with Thyroglobulin in FRTL5 cells. (GRP94는 thyroglobulin의 folding에 관여한다.)

  • Seong, Yeon-Mun;Shong, MinHo;Kwon, O-Yu
    • Journal of Life Science
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    • v.9 no.1
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    • pp.76-83
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    • 1999
  • Mammalial expression vectors containing GRP94, BiP, ERp72, and PDI, were introduced into FRTL5 cells. Transfected cells were selected by neomycin resistance for exogenously overexpressed proteins in the ER. The use of a reducible cross-linker, DSP, markedly improved the ability to detect noncovalent interactions of PDI, BiP and GRP94 with newly-synthesized thyroglobulin. Under normal conditions, GRP94 was found to associate transiently with early Tg folding intermediates, displaying interaction kinetics similar to those reported for another ER chaperones of BiP.

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Pharmacokinetics and Biodistribution in Mice of pCK-VEGF Expressing Human Vascular Endothelial Growth Factor (마우스에서 VEGF발현 Naked DNA 벡터인 pCK-VEGF의 약동력학 및 조직내 분포)

  • 도현미;고준일;이종진;손미원;조홍찬;김종묵;김병문;김선영
    • YAKHAK HOEJI
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    • v.45 no.1
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    • pp.71-77
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    • 2001
  • We recently developed a high efficiency expression vectors pCK, which drives a high level of gene expression in the skeletal muscles of mice. In this study, we investigated the pharmacokinetics and biodistribution of pCK-VEGF expressing human VEGF165 after intravenous or intramuscular administration. The quantity of pCK-VEGF in the tissues of mice was measured by the PCR method which has a detection limit of approximately 1 pg of the exogenously added plasmid. In the case of intravenous administration, the half life of the pCK-VEGF plasmid in the bloodstream was 1.68 min. After intra-muscular administration, the half life of pCK-VEGF plasmid in the bloodstream was 6.78 min. At 90 min post-administration, 30% of the injected pCK-VEGF was found at the site of injection, where it persisted for up to 8 hours. Less than 1.6% of the injected pCK-VEGF plasmid DNA was detected in highly vascularized tissues such as the lung, kidney; and liver at 90 min post-administration, but the plasmid was undetectable at later time points. These results suggested that intramuscularly administrated pCK-VEGF persisted for longer periods of time in muscles than in other tissues and that direct intra-muscular injection of pCK-VEGF might be useful for local therapeutic angiogenesis.

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Expression of Alpha-Amylase Gene from Bacillus licheniformis in Lactobacillus brevis 2.14

  • Lee, Kang-Wook;Park, Ji-Yeong;Kim, Gyoung-Min;Kwon, Gun-Hee;Park, Jae-Yong;Lee, Mee-Ryung;Chun, Ji-Yeon;Kim, Jeong-Hwan
    • Preventive Nutrition and Food Science
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    • v.13 no.3
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    • pp.190-195
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    • 2008
  • The $\alpha$-amylase gene, amyL, from Bacillus licheniformis was expressed in Lactobacillus brevis 2.14 and Escherichia coli $DH5{\alpha}$ using two different shuttle vectors, pCW4 and pSJE. E. coli transformants (TFs) harboring either $pCW4T{\alpha}$ or $pSJET{\alpha}$ produced active $\alpha$-amylase but L. brevis TFs did not, as determined by enzyme assays and zymography. But amyL transcripts were synthesized in L. brevis TFs. In terms of plasmid stability, pSJE, a theta-type replicon, was more stable than pCW4, an RCR (rolling circle replication) plasmid, in L. brevis without antibiotic selection.

A Molecular Biotechnology For Removal of Toxic Heavy Metals

  • Bang Sang-Weon;Clark Douglas S.;Keasling Jay D.
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2000.10a
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    • pp.128-135
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    • 2000
  • The thiosulfate reductase gene (PhsABC) from Salmonella typhimurium was expressed in Escherichia coli in order to produce sulfide from inorganic thiosulfate and precipitate metals as metal sulfide complexes. A 5.1-kb DNA fragment containing the native phsABC and a 3.7-kb DNA fragment, excluding putative promoter and regulatory regions were inserted into expression vectors pTrc99A and pJB866, respectively. Upon expression of phsABC, E. coli DH5$\alpha$ harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strain (E. coli DH5$\alpha$) under both aerobic and anaerobic conditions. Among the four constructs, E. coli DH5$\alpha$ harboring pSB74 produced the highest level of thiosulfate reductase and removed most of heavy metals from solution under anaerobic conditions. In a mixture of 100 $\mu$M each of cadmium, lead, and zinc, the strain could remove $99\%$ of the total metals from solution within 10 hours. Cadmium was removed first, lead second, and zinc last. In contrast, a negative control did not produce any measurable sulfide and removed very little metals from solution. These results have important implications for removal of metals from wastewater contaminated with several metals.

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Efficient Expression System of High Value Proteins, hGM-CSF and hEGF, using Agrobacterium-mediated Chlorella Transformation (아그로박테리움 매개 클로렐라 형질전환을 이용하여 유용 단백질인 hGM-CSF와 hEGF의 효율적인 발현 시스템)

  • Yu Jeong Jeong;Mi ­ Jung Park;Woo ­ Jeong Lee;Sung Chun Kim
    • Journal of Marine Bioscience and Biotechnology
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    • v.16 no.1
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    • pp.26-35
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    • 2024
  • Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

Establishment and Characterization of Immortalized Human Dermal Papilla Cells Expressing Human Papillomavirus 16 E6/E7

  • Seonhwa Kim;Kyeong-Bae Jeon;Hyo-Min Park;Jinju Kim;Chae-Min Lim;Do-Young Yoon
    • Journal of Microbiology and Biotechnology
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    • v.34 no.3
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    • pp.506-515
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    • 2024
  • Primary human dermal papilla cells (HDPCs) are often preferred in studies on hair growth and regeneration. However, primary HDPCs are limited by their reduced proliferative capacity, decreased hair induction potential, and extended doubling times at higher passages. To overcome these limitations, pTARGET vectors containing human papillomavirus16 (HPV16) E6/E7 oncogenes were transfected into HDPCs and selected using G-148 to generate immortalized cells here. HPV16 E6/E7 oncogenes were efficiently transfected into primary HDPCs. Immortalized HDPC showed higher proliferative activity than primary HDPC, confirming an increased proliferation rate. Expression of p53 and pRb proteins was downregulated by E6 and E7, respectively. E6/E7 expressing HDPC cells revealed that cyclin-dependent kinase (CDK) inhibitor p21 expression was decreased, while cell cycle-related genes and proteins (CDK2 and cyclin E) and E2F family genes were upregulated. Immortalized HDPCs maintained their responsiveness to Wnt/β-catenin pathway and hair follicle formation capability, as indicated by their aggregative properties and stemness. E6/E7 immortalized HDPCs may facilitate in vitro hair growth and regeneration studies.