• Korean Journal of Microbiology
• /
• v.35 no.1
• /
• pp.28-34
• /
• 1999

Fragmentation vectors are used to analyze function and genomic structure of a gene of interest by creating deletion derivatives of large fragments of genomic DNA cloned as yeast artificial chromosomes (YACs). Herein, we developed a new hicistronic fragmentation vector that contains internal ribosomal entry sile (IRES) of encephalomyocarditis vin~s (EMCV) and $\beta$-galactosidase as a reporter gene. This vector system provides a novcl loo1 to analyze expression patterns of a gene of interest due to simultaneous expression of a target gene as well as $\beta$-galactosidase driven from a single message. In addition, the bicistronic fragmentation vector contains four rare-cutting restriction enzyme sites in the polycloning sites which can be used to conveniently insert any kinds of genes and therefore facilitates targeting DNA scgments into YAC by means of homologous recombination. This approach establishes a paradigm for manipulation of mammalian DNA segments and characterization of expression and regulatory regions of mammalian gene cloned as YAC.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.7
• /
• pp.1015-1022
• /
• 2007

The spermatogonial stem cell (SSCs) is unique in that it is the only cell in the adult male that can contribute genes to a subsequent generation. Permanent modification of the germ cell line may be realized if stem cells could be cultured, transfected with unique genes, and then transplanted into recipient testes. We developed a culture system that supported long-term viability of SSCs. We used a retrovirus vector (pMSCV including ${\beta}$-galactosidase) to stably transfect spermatogonia following long-term culture using the system developed. Expression of the reporter gene ${\beta}$-galactosidase controlled by the retroviral vector was stable in long-term cultured SSCs. We confirmed the retroviral-mediated ${\beta}$-galactsidase gene could be expressed in germ cells in recipient mice following SSCs transplantation.

• Journal of Microbiology
• /
• v.34 no.1
• /
• pp.74-81
• /
• 1996

Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the .alpha.-amylase gene from an .alpha.-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the .alpha.-amylase gene for easy replacement of various foregn structural genes. To evaluate this secretion vectors, the .betha.-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active .betha.-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each .betha.-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed .betha.- lactamases were located idn the culture medium. The amount of the secreted .betha.-lactamase was about 80% of the total secreted proteins in the culture medium.

• Korean Journal of Plant Resources
• /
• v.27 no.3
• /
• pp.242-248
• /
• 2014

This study was conducted to establish an efficient transformation system by using somatic embryogenesis in an important Korean native conifer, Korean fir (Abies koreana). Embryogenic masses were induced from mature zygotic embryos of the Korean fir on Schenk and Hildebrandt medium, which was supplemented with thidiazuron. For genetic transformation, the embryogenic masses were co-cultivated with a disarmed Agrobacterium tumefaciens strain C58/pMP90 containing the plasmid vector pBIV10 or LBA4404 containing the plasmid vector MP90. Both vectors contain the kanamycin resistance and beta-glucuronidase (GUS) reporter genes. A total of 48 lines of embryogenic masses were selected on mLV medium containing $50{\mu}g/mL$ of kanamycin after 4 weeks of culture, following 3 days of co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 (none of the lines was cultivated with strain LBA4404 carrying MP90). Quantitative real-time PCR was performed, and high levels of GUS transcripts were observed in the 48 putative transgenic lines; however, the control (non-transgenic line) showed negative results. Results of histochemical staining showed that the expression of the GUS reporter gene was observed in somatic embryos that developed from the embryogenic masses of all 48 lines. Stably transformed cultures were successfully produced by co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 in Korean fir. Here, we have reported an Agrobacterium-mediated gene transfer protocol via somatic embryogenesis that may be helpful in developing breeding and conservation strategies for the Korean fir.

• Journal of Aquaculture
• /
• v.17 no.1
• /
• pp.62-68
• /
• 2004

A cDNA encoding the masu salmon, Oncorhynchus masou, estrogen receptor $\alpha$ (msER$\alpha$) was cloned from the pituitary gland by polymerase chain reaction (PCR). This cDNA contains an open reading frame encoding 513 amino acid residues, and the calculated molecular weight of this protein is about 56,430 Dalton. The amino acid sequences of the DNA binding and ligand binding domains of msER$\alpha$ showed high homology to those of other fish species (84-100%). Reverse transcription PCR analysis showed that the mRNA level of msER$\alpha$ in the pituitary was slightly higher in estradiol-17$\beta$(E2) injected masu salmon than that of control fish. To test the biological activity of msER$\alpha$, the cDNA was ligated to a mammalian expression vector and transfected into a gonadotrope-derived cell line, L$\beta$T2, with a reporter plasmid including estrogen responsive element. Expression of the reporter protein, luciferase, was E2 and msER$\alpha$-dependent. The masu salmon ER$\alpha$ is structurally conserved among teleost species and functions as a transcriptional activator in the pituitary cells.

• Korean Journal of Veterinary Research
• /
• v.39 no.4
• /
• pp.772-777
• /
• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2002.05a
• /
• pp.48-50
• /
• 2002

A strategy for development of a functional rice in proved with human lactoferrin and enhancement of nutrient compounds was planned. For the purposes we have cloned and characterized a human lactoferrin cDNA from human mammary gland cDNA library A endosperm storage vacuole targeting sequence and the cDNA fragment was linked to endosperm specific glutelin promoter. The fusion gene fragment was inserted into a binary vector containing MAR gene. In addition a new ${\beta}$-galactosidase gene from Bifidobacterium of human was used as a reporter gene in the vector system, Rice plants showing a high concentration of amino acids in the endosperm cells were developed by using a biochemical mutation and bred for the transformation with the binary vector system Finally we have established a transformation method for the rice endosperm cells.

• Genomics & Informatics
• /
• v.1 no.2
• /
• pp.113-118
• /
• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.18 no.1
• /
• pp.69-76
• /
• 1998

This study was conducted to construct of the transgenic plants wliich are resistant to oxidative stresses including ozone with B. mpestris cytosolic glutathione reductase cDNA using the binary vector system of Agrobacterium tumefaciens. The 1.8kb B. campestris cytosolic GR cDNA was subcloned into the unique Sma I site of the plant transformation vector pBKSI- I, downstream of the constitutive CaMV 35s promoter and upstream of the nos termination sequence, in place of the uidA (GUS) reporter gene. The resulting plant transformation vector, pBKS-GRI, was introduced into A. tumefaciens LBA4404 by two cycles of tkeze-thaw method. The B. nqus cotyledonary petioles were transformed by the Agrubaferium harboring pBKS-GRI. Transformed shoots were induced and selected on regeneration medium supplemented with kanarnycin. The shoot formation was increased remarkably by addition of Ag$NO_3$, in MS media. The transgenic plants were analyzed for the presence of the B. campestris GR gene by Southern blot analysis and it was confirmed that a foregin gene was stably integrated into the genomes of B. nqus plants.

• Food Science of Animal Resources
• /
• v.39 no.1
• /
• pp.13-22
• /
• 2019

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ${\beta}$-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, $pULP3-P_{LDH}$, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.