• Development and Reproduction
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• v.14 no.4
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• pp.215-223
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• 2010

The construction of transgenic mouse using embryonic stem (ES) cells has been crucial in the functional studies of gene on mouse genome. Gene knockout mice have been powerful for elucidating the function of genes as well as a research model for human diseases. Gene targeting and gene trapping mathods have been the representative technologies for making the knockout mice by using ES cells. Since the gene targeting and the gene trapping methods were independently developed about 20 years ago, it's efficiency and productivity has been improved with a advance of molecular biology. Conventional gene targeting method has been changes to high throughput conditional gene targeting. The combination of the advantage of gene targeting and gene tapping elements allows to extend a spectrum of gene trapping and to improve the efficiency of gene targeting. These advance should be able to produce the mutant with various phenotype to target a certain gene, and in postgenome era they have served as crucial research tools in understanding the functional study of whole genome in mouse.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.242-250
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• 2015

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1912-1918
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• 2006

Marine bacterium Hahella chejuensis KCTC 2396 simultaneously produced red antibiotic prodigiosin and undecylprodiginine. A complete set of the prodigiosin biosynthetic gene cluster has been cloned, sequenced, and successfully expressed in a heterologous host. Sequence analysis of the gene cluster revealed 14 ORFs showing high similarity to pig and red genes from Serratia spp. and Streptomyces coelicolor A3(2), respectively, and the gene organization was almost: similar to that of pig genes. These genes were named hap for Hahella prodigiosin, and determined to be transcribed as a single operon, by RT-PCR experiment. Based on the hap gene mutagenesis experiments and comparative analysis with pig and red genes, we propose a prodigiosin-biosynthetic pathway in KCTC 2396.

• BMB Reports
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• v.34 no.6
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• pp.502-508
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• 2001

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

• Journal of Microbiology
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• v.34 no.4
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• pp.327-333
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• 1996

Yeast, like many other microbes, encounters large variations in ambient pH in their natural environments. Microorganisms capable of growing over a wide pH range require a versatile, efficient pH homeostatic mechanism protecting intracellular processes against extremes of pH. In several organisms, fusions to the bacterial lacZ gene have been extremely useful for the identification of genes expressed at different time during the life cycle or under different growth conditions. In this study, using the lacZ gene screening system, we surveyed a large number of yeast strains with lacZ insertion to identify genes regulated by pH. A yeast genomic library was constructed and inserted with lacZ by a shuttle mutagenesis procedure. The yeast transformants were individually picked up with a toothpick, replica-plated, and grown in alkaline pH medium. Among the 35,000 colonies screened, 10 candidate strains were identified initially by the $\beta$-gal assay. We finally confirmed two yeast strains carrying the genes whose expression are strictly dependent on pH of growth medium. One of the fusions showing a 10-fold induction in expression level in response to alkali pH was selected and further characterized. The pH-regulated gene was cloned by inverse PCR and a partial sequence of the gene was determined. Identification and characterization of the gene is currently under investigation.

• BMB Reports
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• v.38 no.1
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• pp.89-96
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• 2005

Earlier, we reported that the bacteriophage $\lambda$ P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this $\lambda$ P gene-mediated lethality. In this paper, we show that under the $\lambda$ P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94% linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from $\lambda$ P gene-mediated killing and complements E. coli dnaAts46 at $42^{\circ}C$. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to $\lambda$ P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.

• Applied Biological Chemistry
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• v.38 no.4
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• pp.313-319
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• 1995

The involvement of the cell-wall degrading enzymes in Rhizobium has long been an unsolved question about the infection process in the formation of root nodule. To assess the contribution of the cellulase to the nodulation of rhzobia, here we report the production of cellulase from R. meliloti TAL1372 which degrade carboxymethylcellulose (CMC) model substrate with CMC-plate method. We constructed a genomic library by cloning Sau3A-digested genomic DNA from R. meliloti TAL1372 into the BamHI site of the cosmid vector pLAFR3. Out of more than one thousand transductants of E. coli, one clone (pRC8-71) had CM-cellulase activity and contained pLAFR3 cosmid with 30 kb insert of R. meliloti DNA The product of CM-cellulase gene was analyzed by native PAGE. About 45 kD protein was considered to be a product of the gene. Tn5 mutagenesis reveals that the structural gene located in a ca. 3 kb KpnI fragment. The cellulase-minus mutants of R. meliloti TAL1372 were obtained by Tn5 mutagenesis of pRC8-71 and marker exchange techniques. Analyses of the nodulation ability of these Tn5 mutants showed that the CM-cellulase gene of R. meliloti TAL1372 may be involved in early nodulation development on alfalfa (Medicago satiua).

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.96-96
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• 2002

상동유전자 재조합기술을 myostatin 유전자에 적용하기 위해 돼지 골격에 붙어 있는 근육으로부터 RNA를 추출하였고 돼지 Myostain Exon 3 부위의 specific primer를 제작하여 RT-PCR 을 수행 한 후 증폭된 342bp DNA 를 추출하여 T vector 에 ligation한 후 sequencing을 실시하여 돼지 genomic DNA 에서 Myostatin gene 의 Exon 3 부위와 100% match 되는 것을 확인하였다. (omitted)

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2002.10a
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• pp.183-184
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• 2002

The use of transgenic fish has so far been chiefly limited by the lack of predictable, strong, tissue specific, and position-independent expression of transgenes. For genetic analysis, expression of a marker transgene, easily screenable in the living fish, could facilitate studies of gene targeting, insertional mutagenesis, lineage, and mutational analysis. (omitted)

• Korean Journal of Microbiology
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• v.26 no.1
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• pp.20-26
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• 1988

Three $NIf^{-}$ -mutants were isolated from Enterbacter agglomerans 339 through the transposon umtagenesis using a RP4-mobilising system for its nif-gene characterization. All mutants hadn't acetylene-reduction ability. Then we confirmed that Tn5 was inserted into all conserved nif-plasmids through the Southern Hybridization.