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Efficient Gene Targeting using Nuclear Localization Signal (NLS) and Negative Selection Marker Gene in Porcine Somatic Cells

  • Kim, Hye Min;Lee, Sang Mi;Park, Hyo Young;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.38 no.2
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    • pp.71-77
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    • 2014
  • The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

Developing a Gene-trapping Approach for Gene Identification Using Nuclear Transfer in Zebrafish (지브라물고기 복제방법에 의한 유전자 동정 및 유전자트랩법 개발)

  • Lee, K.Y.
    • Journal of Animal Science and Technology
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    • v.46 no.2
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    • pp.155-164
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    • 2004
  • This involves identifying and cloning trapped genes from cultured cells carrying the gene-trap constructs and generating cloned zebrafish using these cells for functional study. Gene-trapping studies in gene-trapped cells were carried out in initial and cloned zebrafish carrying gene-trap events were successfully produced based on the nuclear transplantation technique. Two kind of retroviral gene-trap constructs were adopted. The first one(SA/GFP-TP), constructed in my laboratory, carries a GFP reporter gene containing a splicing acceptor and an internal neo gene. The second one(Neo-TP), obtained from Dr. Hicks (Hicks et al., 1997), contains a promoter-less neo gene located in the LTR sequence of a retroviral vector. The infected cells were subjected to drug selection(neomycin treatment) because the two constructs carry the neomycin resistant gene. All those cells survived the neomycin treatment should carry the proviral insertions. For Neo-TP, Isolated DNA from the neomycin-resistant fibroblast cells infected by Neo-TP, was digested with EcoR1 restriction enzyme and transformed into bacteria after ligation. This procedure led to the isolation of seven clones carrying flanking cellular DNA with a typical retroviral integration signature sequence. These clones contained genomic DNA ranging from 1kb to 7kb and sequences of 300-600 bp were obtained from each of the rescued plasmids. Database searching showed that all of them share high homology to zebrafish sequences. For fish cloning using tagged cells, initially, nucleus donors directly selected from a mixture of cells(Neo-TP cells) were used. A total of 44 embryos(3.7%) out of 1179 transplants were reached blastula stage; 8 of these embryos(0.8%) hatched and 3(0.3%) of them survived to adulthood. One out of three lived cloned zebrafish has an amplified fragment and was labeled with 32P.

A Study of the foundations of culture established the Sosu Seowon (소수서원(紹修書院) 건립의 문화적 토대 연구 - 회헌(晦軒) 안향(安珦)의 선비정신을 중심으로 -)

  • Lee, Jeong-Hwa
    • (The)Study of the Eastern Classic
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    • no.48
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    • pp.41-63
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    • 2012
  • This research paper is aims to study the Zhu Xi school of Neo-Confucianism culture based on the Sosu seowon. Also This research paper is to discuss study and scholars correlation the Sosu seowon. The Sosu seowon is mainly made up of the Zhu Xi school of Neo-Confucianism development. The Sosu seowon was the cradle of scholars and bureaucrats. Scholars who study of orthodox Neo-Confucianism in the our country was respected academic spirit of An Whyang in the study of orthodox Neo-Confucianism. An Whyang is symbolic existence of the Sosu seowon. He knew well the importance of learning. He was the person to represent the Sosu seowon. Toegye and Sinje are handed down to the fundamental principle brought in on the orthodox Neo-Confucianism caused by An Whyang. This research paper is to based upon on academic spirit of An Whyang in the study of orthodox Neo-Confucianism. Scholars who study of orthodox Neo-Confucianism thought that the nation's happiness made appointment to a competent person learned the orthodox Neo-Confucianism caused by An Whyang related to the Humanism education.

A Study on the Neo-Avantgarde Tendency Expressed in the Modern Make-up -with the Main Point of the Late 1990's Catwalk- (현대 메이크업에 나타난 네오아방가르드 경향에 관한 연구 -1990년대 후반 캣워크를 중심으로 -)

  • 장미숙;양숙희
    • The Research Journal of the Costume Culture
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    • v.7 no.3
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    • pp.72-92
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    • 1999
  • The purpose of this thesis is to review relationship between various expression method in modern make-up and the tendency of Neo-Avantgarde. In this paper, the author classified the distinctive characters in Neo-Avantagarde with Historicism, Decadence, Humor, and Inhumanism and explained innovation and creatio which is manifested in the late 1990\`s make-up by studying Neo-Avantgarde tendency expressed in modern make-up. The results are as follows. 1. The Historicism in modern make-up is expressed in Japanese Kabuki Make-up, Chinese Pecking Opera make-up, and Primitive mask Make-up in Africa. 2. The Decadence is manifested in Tattoo make-up which is expressing fetishism and fin-de-냗칟 image, and Snobism Make-up which is expressing selg-ostentation and eroticism with artificial nail and eyelash. 3. The humor appears to the Character Make-up copying the characters in doll plays, animation and juvenile story. In addition it is showed in Kitsch Make-up, which has vulagr, rustic and childish image by wearing so diffusive and cheap ornaments. 4. The Inhumanism is appeared in Graphic Make-up which is in the pursuit of the visual happiness. It includes simple geometrical type, drawing and coloring. It is also showed in Vampire Make-up, which means the make-up style mading horro, hizarrerie, and mistery by primary color(black and red) make-up. As the above, Neo-Avantgarde can account for the diversity and pluralism of contemporary make-up.

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