• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.564-570
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• 2016

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using $300{\mu}g/mL$ G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

• 미생물학회지
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• 제32권4호
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• pp.264-270
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• 1994

세균의 RNA 중합효소에서 여러 ${\sigma}$ 인자들 간에 보존된 아미노산 서열중 2.3 부위와 4.2 부위의 아미노산 서열로부터 유 n하여 두가지의 PCR primer를 제작하였다. 이들을 이용하여 PCR을 수행하였을 때, E. coli와 Streptomyces coelicolor의 DNA로부터 예상되었던 480 bp 정도의 DNA가 증폭되는 것을 관찰하였다. E. coli DNA에서 증폭된 DNA를 클로닝하여 염기서열을 결정한 결과 E. coli의 rpoS 유전자로부터 유래하였음을 알았다. 이를 탐침으로 S. coelicolor에서 genomic DNA hybridization을 수행하였을 때, PvuII 절편 두가지 (3.5 kb, 2.0 kb) 와 SalI 절편 두가지(3.4kb, 1.5 kb)에 탐침이 결합하는 것을 관찰하였다. 3.5 kb의 pvuII 절편을 sublibrary로부터 클로닝하고, 탐침이 결합하는 1.0kb의 BamHI/HincII 절편의 염기서열을 분석하였다. 부분적으로 결정된 염기서열을 BLAST 프로그램을 이용하여 GenBank와 EMBL, PDB 등의 data library의 유전자들과 비교하여 본 결과Streptomyces속의 ${\sigma}$인자들을 비롯한 Synechococcus종, Anabaena종, Pseudomonas aeruginosa, Stigmatella aurantica 등의 주된 ${\sigma}$ 인자와 높은 유사성을 보였다. 현재까지 1.2 부위와 4 부위에 해당하는 부분의 염기서열을 결정하였는데, 이 부분은 S. coelicolor에서 알려진 다섯가지의 ${\sigma}$ 인자 유전자 중 hrdA와 가장 높은 유사성을 보이며, 아미노산의 유사성이 1.2부위에서는 88%, 4 부위에서는 75%인 것으로 나타났다.

• BMB Reports
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• 제51권7호
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• pp.338-343
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• 2018

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.