• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1279-1287
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• 2012

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, $10^{-5}-10^{-3}\;recipient^{-1}$). pSTE33T showed lower efficiency ($10^{-7}-10^{-6}\;recipient^{-1}$) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.111-111
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• 2005

• Bulletin of the Korean Chemical Society
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• v.32 no.8
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• pp.2599-2606
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• 2011

The fluorescence of ethidium bound to DNA, poly[d(A-T)$_2$], and poly[d(G-C)$_2$] at a [ethidium]/[DNA] ratio of 0.005 was quenched by porphyrins when both ethidium and the porphyrins simultaneously bound to the same polynucleotide. The quenching was tested using the "inner sphere" and the "Forster resonance energy transfer" (FRET) models, with the latter found to contribute, at least in part, to the quenching. Meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) exhibited a higher quenching and FRET efficiency than cis-bis(N-methylpyridinium-4-yl)porphyrin (BMPyP) for all of the tested DNA and polynucleotides, demonstrating that energy transfer efficiency is affected by the number of positive charges of porphyrins.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

• Bulletin of the Korean Chemical Society
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• v.32 no.9
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• pp.3223-3228
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• 2011

To quantify the presence of mercuric ions in aqueous solution, double-stranded DNA (dsDNA) of poly(dT) was employed using a light switch compound, $Ru(phen)_2(dppz)^{2+}$ (1) which is reported to intercalate into dsDNA of a right-handed B-form. Addition of mercuric ions induced the dehybridization of poly(dT)${\cdot}$poly(dA) duplexes to form a hairpin structure of poly(dT) at room temperature and the metal-to-ligand charge transfer emission derived from the intercalation of 1 was reduced due to the dehybridization of dsDNA. As the concentration of $Hg^{2+}$ was increased, the emission of 1 progressively decreased. This label-free emission method had a detection limit of 0.2 nM. Other metal ions, such as $K^+$, $Ag^+$, $Ca^{2+}$, $Mg^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Ni^{2+}$, $Cu^{2+}$, $Cd^{2+}$, $Cr^{3+}$, $Fe^{3+}$, had no significant effect on reducing emission. This emission method can differentiate matched and mismatched poly(dT) sequences based on the emission intensity of dsDNA.

• Bulletin of the Korean Chemical Society
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• v.26 no.4
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• pp.537-542
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• 2005

It was proposed that Ru(II)[(1,10-phenanthroline)$_2$dipyrido[3,2-a:2',3'-c]phenazine ([Ru(phen)$_2$DPPZ]$^{2+}$)complexes and 4',6-diamidino-2-phenylindole (DAPI) simultaneously bind to poly[d(A-T)$_2$] (Biophysics. J. 2003, 85, 3865). Förster type resonance energy transfer from excited DAPI to [Ru(phen)2DPPZ]$^{2+}$ complexes was observed. In this study, we synthesized $\Delta$- and $\wedge$-[Ru(phenanthroline)$_2$dipyrido[3,2-a:2’3’c]6-azaphenazine] ([Ru(phen)$_2$DPAPZ]$^{2+}$) at which the DNA intercalating ligand DPPZ was replaced and we studied its binding properties to poly[d(A-T)$_2$] in the presence and absence of DAPI using polarized spectroscopy and fluorescence techniques. All the spectroscopic properties of the [Ru(phen)$_2$DPAPZ]$^{2+}$-poly[d(A-T)$_2$] complex were the same in the presence and absence of DAPI that blocks the minor groove of polynucleotide, suggesting both $\Delta$- and $\wedge$-[Ru(phen)$_2$DPAPZ]$^{2+}$ complexes are located at the major groove of poly[d(A-T)2]. On the other hand, in contrast with [Ru(phen)$_2$DPPZ]$^{2+}$, both $\Delta$- and $\wedge$-[Ru(phen)$_2$DPAPZ]$^{2+}$ exhibited almost twice the efficiency in the fluorescence quenching of DAPI that binds at the minor groove of poly[d(A-T)$_2$]. This observation indicates that the efficiency of the Förster type resonance energy transfer can be controlled by a small change in the chemical structure of the intercalated ligand.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.4
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• pp.520-524
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• 1999

The present experiments were designed to examine whether exogenous DNA injected into the germinal crescent region (GCR) of early stage of developing embryos, which is considered to be the main place from which PGCs originate, can be transferred to recipient chicken embryos. In this experiment, Miw Z (DNA) dissolved in the transfection reagent (TR: Boehringer, Germany) was introduced into the GCR of donor embryos at stage 3-5 or 9-11, followed by continued incubation until the stage 13-15 of embryonic development. The PGCs collected from the embryonic blood vessels were examined for the incorporation of the injected DNA into the PGCs by the methods of X-gal staining and PCR analysis. As the results, the foreign DNA was successfully incorporated into the PGCS, leading to their transfer to the gonadal tissues. The present results, therefore, suggest that the early stage (3-5 or 9-11) of chicken embryonic development would be more successful than stage 13-15 in transferring exogenous genes to the recipient embryos, leading to the possibility of producing transgenic chicken medianting the PGCS.

• Progress in Superconductivity and Cryogenics
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• v.19 no.1
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• pp.9-12
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• 2017

Recently, DNA vaccination is attracting attentions as a new therapeutic method for lifestyle diseases and autoimmune diseases. However, its clinical applications are limited because a safe and efficient gene transfer method has not been established yet. In this study, a new method of gene transfer was proposed which utilizes the jet injection and the magnetic transfection. The jet injection is a method to inject medical liquid by momentary high pressure without needle. The injected liquid diffuses in the bio tissue and the endocytosis is considered to be improved by the diffusion. The magnetic transfection is a method to deliver the conjugates of plasmid DNA and magnetic particles to the desired site by external magnetic field. It is expected that jet injection of the conjugates causes slight membrane disruptions and the traction of the conjugates by magnetic field induces the efficient gene transfer. In conclusion, the possibility of improvement of the gene expression by the combination of jet injection and magnetic transfection was confirmed.

• Mycobiology
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• v.29 no.3
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.

• Current Research on Agriculture and Life Sciences
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• v.11
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• pp.11-17
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• 1993

The transfer of genetic material into pea tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens containing the binary vector. The method used for transformation requires non-tissue culture steps as it involves the inoculation of the site of the shoot removed of germinating seeds. The identification of ${\beta}$-glucuronidase activity in the tissues of $T_0$ pea plants indicates that the plant expressible ${\beta}$-glucuronidase gene, contained the T-DNA region from pLPBO2, had been transferred at least into somatic tissues. Putative transformed $T_0$ pea plants were advanced to produce $T_1$ plants which were also assayed for the presence of the transferred ${\beta}$-glucuronidase gene. The presence of the ${\beta}$-glucuronidase gene in DNAs isolated from $T_1$ plant was demonstrated by DNA gel blot hybridization. This analysis revealed that the transformed plants contained ${\beta}$-glucuronidase gene.