• Journal of fish pathology
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• v.22 no.3
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• pp.375-382
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• 2009

Integration and expression of a target gene into chromosomal genomes of host cell by retrovirus mediated gene transfer system usually require complicate and laborious procedures. In the present study, we investigate a simple method to integrate a target gene into genome of BF-2 cells using ultraviolet (UV)-inactivated snakehead retrovirus (SnRV), a fish retrovirus. First of all, an optimization of transfection condition was determined with BF-2 cells using Lipofectamine 2000 and Transome. Using 0.5 $\mu\ell$ Lipofectamine 2000 resulted in 33.8, 40.6 and 40.2% of transfection efficacy with high survival rate (minimum 80%) in 0.5, 1 and 2 $\mu{g}$ DNA, respectively, and those of Transome were all less than 5%. It was confirmed that UV-treatment for 5 min was enough to inactivate infectivity of SnRV. Next, a cassette composed of GFP (green fluorescent protein) gene flanked by LTR (long terminal repeats) sequences derived from SnRV was constructed and transfected into BF-2 cells followed by treatment with UV-inactivated SnRV for optimization of integration and expression of the cassette gene. As the results, the fluorescence was expressed in BF-2 cells treated with UV-inactivated SnRV 3 and 5 times, while there was no expression in BF-2 cells with once and non treatment. Accordingly, it was confirmed that GFP gene was integrated into chromosomal genome of BF-2 cells with UV-inactivated SnRV.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.110-115
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• 2016

A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas - Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.13-18
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• 1999

The totipotential spermatogonial stem cells of adult testis which give rise to mature sperm cells is well-known to incorporate foreign DNA as well as those of somatic cells. Also, the integration rates of foreign DNA after haploid stages are generally known to decrease and /or is simply bound foreign DNA into the sperm plasma membrane. To overcome these problems, liposome and DNA complexes were used to determine how direct injection of these complexes into testis were integrated into sperm genome and resulted in transgenic offspring. To study this purpose, cation liposome was gently mixed with WAF/hGH DNA (1 : 2) and the complexes were injected into testis. At 10, 20, 40, 60, and 80 days after direct injection into testis, mature sperm cells were recovered by using artificial virgin method from two goats and each semen except a part of semen used for DNA analysis such as PCR or Southern blotting was cryopreserved for the artificial insemination. The results obtained in this study are summarized as follows. 1. By PCR, the presence of exogenous DNA was confirmed up to 80 days after injection with liposome/DNA complexes. The highest integration rates were obtained at day 40 after direct injection. This results suggested that spermatogonial stem cells were integrated exogenous DNA into their genome. 2. Among 23 Korean Native Goats which were artificially inseminated, 4 goats resulted in pregnancy and produced 7 young goats. 3. Two young goats were confirmed as a transgenic by PCR and Southern blot analysis. Therefore, our results suggested that testis-mediated gene transfer can be used as a feasible tools for the production of transgenic livestock.

• Korean Journal of Microbiology
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• v.31 no.1
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• pp.48-53
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• 1993

For the purpose to improve the glucoamylase productivity of Saccharomyces diastaticus, we integrated STA 1 gene into chromosomal DNA of S. diastaticus using YIp vector. After construction of Ylp-STA by the subcloning of STAI (5.3 kb) into YIp5 vector, S. diastaticus GMT-II(a. ura3. STAJ) was transformed by Ylp-STA through homologous recombination at the chromosomal STAJ gene. So we obtained the tram formants that glucoamylase productivity was increased maximum six fold. These strains transformed by the multi-copy integration of Ylp-STA in chromosomal DNA were confirmed by Southern hybridization. And the integrated Ylp-STA was maintained stably during 30 mitotic divisions.

• Korean Chemical Engineering Research
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• v.47 no.5
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• pp.538-545
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• 2009

The principle and application of a scanning probe microscopy(SPM) are reviewed briefly, and a low-invasive single cell manipulation and a gene delivery technique using an etched atomic force microscopy(AFM) probe tip, which we call a nanoneedle, are explained in detail. The nanoneedle insertion into a cell can be judged by a sudden drop of force in a force-distance curve. The probabilities of nanoneedle insertion into cells were 80~90%, which were higher than those of typical microinjection capillaries. When the diameter of the nanoneedle was smaller than 400 nm, the nanoneedle insertion into a cell over 1 hour had almost no influence on the cell viability. A highly efficient gene delivery and a high ratio of expressed gene per delivered DNA compared the conventional major nonviral gene delivery methods could be achieved using the gene modified nanoneedle.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.266-266
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• 1994

$B^{30}$ 위치에 homoserine이 치환된 사람 insulin 유사체 ($B^{30}$ -homoserine) insulin을 생산하기 위해, insulin의 B 사슬 유전자에 A 사슬 유전자를 직접 연결한 insulin 유전자를 설계하였다. 이 유전자는 10개의 oligonucleotide로 나누어 합성하여 T4 DNA ligase로 결합시킨 후, pUC19 plasmi의 polylinker 영역에 삽입하였다. 이 유전자의 발현을 높이기 위해 이 유전자는 다시 tac promoter의 지배를 받는 lacZ 유전자의 Cia I 또는 Hpa I 제한부위에 도입하여 융합시켰다. 이렇게 구축된 운반체 pTBA나 pKBA를 Escherichia coli JM103 균주에 형질도입시킨 후, 이를 4시간 배양한 후 0.05mM이상의 isopropyl-$\beta$-D-thiogalactopyranoside (IPTG)를 배지에 공급해 주고 2시간 더 배양하였을 때 유전자 발현이 잘 유도되어짐을 알 수 있었다. 이 때 생산된 insulin 전구체들은 세포내 불온성인 inclusion body로 축적되어지는 것을 관찰하였으며, 그 생산량은 세포내 전체 단백질량의 30%에 달하였다.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.45-51
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• 1991

Inserting the $cI_{857}$ structural gene in the downstream of the tac promoter for the overproduction of $cI_{857}$ repressor protein was studied. DNA fragment containing $cI_{857}$ ; repressor gene was amplified by using plasmid pUC12, and partially digested with HphI. Only the $cI_{857}$ structural gene isolated was inserted in the downstream of the tac promoter. Plasmid pDR540- $cI_{857}$ having the tuc promoter-$cI_{857}$ structural gene insert could be isolated by the immunity of cells resistant at $30^{\circ}C$ and cell lysis at $42^{\circ}C$ to $\lambda$ phage $cI_{90}$. The amount of $cI_{857}$ repressor as 17% of total cellular protein were produced by using general E. coli as well as $lacI^q$ JM103 having this plasmid.

• Journal of Life Science
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• v.21 no.3
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• pp.459-463
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• 2011

The genome of the rhesus monkey has diverged as an average sequence identity of ~93%. The rhesus monkey has been widely used as a non-human primate in the field of biomedical and evolutional research. Insertion of transposable elements (TEs) induced several events such as transcriptional diversity and different expression in host genes. In this study, 112 transcripts were identified from a full-length cDNA library of brain tissues of the rhesus monkey. One transcript (R54) showed a different expression pattern between human and rhesus monkey tissues. This phenomenon can be an explanation that R54 transcript was acquired by splicing a donor site derived from exonization of the L2A element. Therefore, integration of TEs during primate radiation could contribute to transcriptional diversity and gene regulation.

• Journal of Aquaculture
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• v.11 no.4
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• pp.457-463
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• 1998

An expression vector, pUC19N6-luc, containing nuclear matrix attachment region(MAR) isolated from Misgurnus mizolepis liver and control expressino vector, pUC19-luc, were constructed. After these vectors were transferred into CHSE-214 cell line by electroporation, the expression rate of luckferase gens, copy number of vectors and chromosome integration of vectors were analyzed by using assay of luciferase activity, PCR and Southern blotting. While the expression pattern of luciferase gene of pUC19-luc was shown in typicla transient ecpression pattern, that of pUC19N6-luc was highly increased at the 5 days after transfectrion. Although the cope number of pUC19N6-luc vector was higher than that of pUC19-luc vector, these vectors were integrated into chromosome at the same time point in the transfected CHSE-214 cells. In conclusion, the increase of luciferase gene expression of pUC19N6-luc was resulted from not the maintaining of the high copy number but the formation of transcription-favorable structure by MAR effect after chromosomal integration.

• Korean journal of applied entomology
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• v.35 no.4
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• pp.321-325
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• 1996

To isolate a novel gene for antibacterial peptide, an inducible clone(BmInc8) was selected by differential screening strategy from Bombyx mori cDNA library prepared from lavae injected with Escherichia coli. This clone contained a cDNA insert of 564 nucleotides and encoded 59 amino acids with an apparent molecular mass of 6.3 kDa. The cDNA sequence of BmInc8 had 61.2% identity compared to that of the bactericidin from Manduca sexta and also the deduced amino acids sequences from this insert had 65% identity compared to that of the cecropin D peptide Hyalophora cecropia. The transient expression assay of this insert using prokaryotic expression vector system revealed that the expressed peptide displayed the antibacterial activity. The cDNA sequence was deposited in GenBank under the accession number U30289.