• YAKHAK HOEJI
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• v.31 no.2
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• The Microorganisms and Industry
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• v.18 no.3
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• pp.2-9
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• 1992

이 논문에서는 대사공학에의 응용에 필수적이며 또한 그 자체의 기술이 학문적으로 상당히 관심을 끄는 C. acetobutylicum에서의 primary metabolic gene cloning에 대하여 정리해 보고자 한다. 우선 C. acetobutylicum의 primary metabolism과 일반적인 대사 조절에 대하여 간략히 살펴보고 이에 관여한 효소들과 gene cloning에 대하여 기술하고자 한다.

• Journal of Life Science
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• v.16 no.6
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• pp.1036-1043
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• 2006

The transformation-associated recombination (TAR) cloning technique allows selective isolation of chromosome regions or genes from complex genome. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosome region of interest. This method involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). To examine whether TAR cloning can be applied to the isolation of gene homologues, we chose the HPRT genes from human and mouse genome. As results, the yield of positive clones for HPRT gene from human and mouse genome when using a TAR vector containing mHPRT hook or hHPRT hook was almost same level. Analysis of the gap regions in mHPRT revealed that they contain abnormalities that could result in instability of the sequences. In conclusion, we were able to use the TAR cloning technology to isolate gene homologue (orthologue) from nonidentical genome. Moreover, the use of the TAR cloning system may accelerate work on closing the remaining gaps in mammalian genome to achieve the goal of annotation of all mammalian genes.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.67-76
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• 2008

Since the birth of Dolly using fully differentiated somatic cells as a nuclear donor, viable clones were generated successfully in many mammalian species. These achievements in animal cloning demonstrate developmental potential of terminally differentiated somatic cells. At the same time, the somatic cell nuclear transfer (SCNT) technique provides the opportunities to study basic and applied biosciences. However, the efficiency generating viable offsprings by SCNT remains extremely low. There are several explanations why cloned embryos cannot fully develop into viable animals and what factors affect developmental potency of reconstructed embryos by the SCNT technique. The most critical and persuasive explanation for inefficiency in SCNT cloning is incomplete genomic reprogramming, such as DNA methylation and histone modification. Numerous studies on genomic reprogramming demonstrated that incorrect DNA methylation and aberrant epigenetic reprogramming are considerably correlated with abnormal development of SCNT cloned embryos even though its mechanism is not fully understood. The SCNT technique is useful in cloning farm animals because pluripotent stem cells are not established in farm animal species. Therapeutic cloning combined with genetic manipulation will help to control various human diseases. Also, the SCNT technique provides a chance to overcome excessive demand for the organs by production of transgenic animals as xenotransplantation resources. Here, we describe the factors affecting the efficiency of generating cloned farm animals by the SCNT technique and discuss future directions of animal cloning by SCNT to improve the cloning efficiency.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1481-1483
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• 2010

We constructed an efficient T-vector, pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones owing to insertional inactivation of the esterase reporter. Additionally, PCR products were efficiently cloned into this vector without the gel purification steps, owing to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.34-39
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• 1994

The gene encoding ${\alpha}$-amylase of Schwanniomyces castellii was cloned in Saccharomyces cerevisiae. The 5.0-kilobase insert was shown to direct the synthesis of ${\alpha}$-amylase. Southern blot analysis confirmed that this ${\alpha}$-amylase gene was derived from the genomic DNA of Sch. castellii. Immunoblot analysis showed that ${\alpha}$-amylase production from S. cerevisiae transformant was less than that of donor strain. The ${\alpha}$-amylase secreted from S. cerevisiae transformant was shown to be indistinguishable from that of Sch. castellii on the basis of molecular weight and enzyme properties.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1123-1126
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• 2011

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.29-48
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• 2003

It is remarkable that nuclear transfer using differentiated donor cells can produce physiologically normal cloned animals, but the process is inefficient and highly prone to epigenetic errors. Aberrant patterns of gene expression in clones contribute to the cumulative losses and abnormal phenotypes observed throughout development. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. These issues raise animal welfare concerns that currently limit the acceptability and applicability of the technology. It is expected that improved reprogramming of the donor genome will increase cloning efficiencies realising a wide range of new agricultural and medical opportunities. Efficient cloning potentially enables rapid dissemination of elite genotypes from nucleus herds to commercial producers. Initial commercialization will, however, focus on producing small numbers of high value animals for natural breeding especially clones of progeny-tested sires, The continual advances in animal genomics towards the identification of genes that influence livestock production traits and human health increase the ability to genetically modify animals to enhance agricultural efficiency and produce superior quality food and biomedical products for niche markets. The potential opportunities in animal agriculture are more challenging than those in biomedicine as they require greater biological efficiency at reduced cost to be economically viable and because of the more difficult consumer acceptance issues. Nevertheless, cloning and transgenesis are being used together to increase the genetic merit of livestock; however, the integration of this technology into farming systems remains some distance in the future.