In vitro Selection of the 2'-Fluoro-2'-Deoxyribonucleotide Decoy RNA Inhibitor of Myasthenic Autoantibodies

Seo, Hwa Seon,Lee, Seong Wook 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.5

Myasthenia gravis (MG) is caused mainly by autoantibodies directed against acetylcholine receptors located in the postsynaptic muscle cell membrane. Using in vitro selection techniques, we isolated an RNA containing 2'-fluoro pyrimidines that can specifically and avidly (K_d∼25nM) bind rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the acetylcholine receptors. This RNA can act as a very effective decoy and block mAb198 binding to the receptors in vitro. Furthermore, this RNA decoy can prevent the antigenic modulation of the acetylcholine receptor caused by mAb198 in human muscle cell cultures with an IC_50 of approximately 2.4μM. These results indicate that the RNA selected in this study is a more potent decoy inhibitor of myasthenic antibodies than the previously identified RNA with 2'-amino pyrimidines [11]. Moreover, this RNA cross-reacts with autoantibodies from patients with MG and can protect human cells from the effects of these antibodies. These observations have important implications for developing an antigen-specific treatment of autoimmune diseases including MG, which is based on decoy RNAs selected in vitro.

In Vitro Selection of Hammerhead Ribozymes with Optimized Stems I and III

Lee,Younghoon,Sim,Soyeong,Kim,Semi,Kim,Hadong,Park,Inwon,Ahn,Jeong Keun,Cho,Bongrae The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.2

A pool of cis-acting bammerhead ribozymes randomized in their substrate recognition sequences was constructed A variety of active cis-acting ribozymes which had various structures of stems Ⅰand Ⅲ was selected from the pool by in vitro selection. The selected ribozymes were cloned and sequenced. The relationship between the cleavage efficiency and basepairing in stems Ⅰ and Ⅲ of the selected ribozymes was investigated. The ribozymes with the smaller difference in folding energies between the active conformation and the stable but inactive conformation showed a tendency to have the batter cleavage efficiency. The optimum length of stem Ⅰ was 5 or 6 bases while the longer stem Ⅲ, in general, appeared to be required for efficient cleavage. The specificity of the ribozyme reaction is discussed in terms of the length of stems Ⅰ and Ⅲ.

In Vitro Selection of Groundnut Cell Lines from Cercosporidium Personatum Culture Filtrates and Regeneration of Resistant Plants Through Cell Culture

Venkatachalam, P.,Geetha, N.,Jayabalan, N.,Saravanababu, S. 한국식물학회 1998 Journal of Plant Biology Vol.41 No.4

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence of Cercosporidium personation pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25-100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced by C. personatum. Selected cell lines were used for plant regeneration on regeneration media containing C. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R_2 generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants through in vitro selection.

In Vitro Selection of Hammerhead Ribozymes with Optimized Stems I and III

Sim, So-Yeong,Kim, Se-Mi,Kim, Ha-Dong,Ahn, Jeong-Keun,Lee, Young-Hoon,Cho, Bong-Rae,Park, In-Won Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.2

A pool of cis-acting hammerhead ribozymes randomized in their substrate recognition sequences was constructed. A variety of active cis-acting ribozymes which had various structures of stems I and III was selected from the pool by in vitro selection. The selected ribozymes were cloned and sequenced. The relationship between the cleavage efficiency and base-pairing in stems I and III of the selected ribozymes was investigated. The ribozymes with the smaller difference in folding energies between the active conformation and the stable but inactive conformation showed a tendency to have the better cleavage efficiency. The optimum length of stem I was 5 or 6 bases while the longer stem III, in general, appeared to be required for efficient cleavage. The specificity of the ribozyme reaction is discussed in terms of the length of stems I and III.

SELEX: Combinatorial Library Methodology and Applications

김성건,Ben C. Shin,Taylor A. Ott,차원석,라임정,윤문영 한국바이오칩학회 2008 BioChip Journal Vol.2 No.2

The SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology is utilized to find aptamers, target-binding oligonucleotides, by <i>in vitro</I> selection. A wide variety of methodologies would be utilized to obtain the aptamers from the randomly degenerate production of oligonucleotides. An aptamer can be modified to have high specificity and affinity to their target. For example, single-stranded oligonucleotides (ssDNA or RNA) and double-stranded oligonucleotides (dsDNA) have been used to select aptamers as anti-infectional drugs or sensitive disease-detecting materials. The aptamers permit chemical structure modifications which enhance their stability and provide better applications. SELEX would be a promising future biotechnology for developing therapeutic and diagnostic agents.

CD44 targeting biocompatible and biodegradable hyaluronic acid cross-linked zein nanogels for curcumin delivery to cancer cells: <i>In vitro</i> and <i>in vivo</i> evaluation

Seok, Hae-Yong,Sanoj Rejinold, N.,Lekshmi, Kamali Manickavasagam,Cherukula, Kondareddy,Park, In-Kyu,Kim, Yeu-Chun Elsevier 2018 Journal of controlled release Vol.280 No.-

<P><B>Abstract</B></P> <P>In this study, we developed novel hyaluronic acid cross-linked zein nanogels (HA-Zein NGs) to deliver the potential anticancer agent curcumin (CRC), a naturally occurring phytochemical drug in cancer cells. <I>In vitro</I> studies showed that they are highly compatible with the tested cell lines. They showed CD44 specific uptake in CT26 cell line more than by the CD44 receptor pre-inhibited CT26 cells. The CRC encapsulated HA-Zein NGs (HA-Zein-CRC NGs) found to exert a specific toxicity against CT26 sparing healthy normal fibroblast cells <I>in vitro</I>. The apoptotic effects were further confirmed with flow cytometry showing that the HA-Zein-CRC NGs exhibited high anticancer activity against the CT26 cells. The <I>in vivo</I> bio-distribution with a CT26 tumor model showed their high tumor accumulation thereby improved antitumor efficacy with a low dosage of CRC, compared to the previous reports. Thus, the preclinical studies clearly showed that these novel HA-Zein NGs would be highly beneficial in encapsulating hydrophobic drugs with improved pharmacokinetics thereby enhancing the therapeutic outcomes.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins

Park, J.H.,Kwon, H.W.,Jeong, K.J. Society for Bioscience and Bioengineering, Japan ; 2013 Journal of bioscience and bioengineering Vol.116 No.2

Owing to simple mechanism of linking and efficient display of proteins, plasmid display system in which proteins are physically linked to plasmids, has been considered as a promising emerging tool in protein engineering. We used human Oct-1 DNA-binding domain (DBD) which can bind to octameric DNA sequence (5'-ATGCAAAT-3') with high affinity, as a potential anchoring motif for plasmid display system. Using three model proteins, histidine hexamer (His<SUB>6</SUB>), glutathione S-transferase (GST) and antibody fragment, we confirmed that Oct-1 DBD fused proteins were strongly linked to plasmids and their linking were conserved for entire process of in vitro selection. Also, the feasibility of this display system was examined using several enrichment experiments from binary libraries. Using Oct-1 plasmid display system, the GST-displayed plasmids were successfully enriched 8500-fold from a large excess (10<SUP>4</SUP> fold) of negatives (non-GST plasmid). From the results, Oct-1 DBD-based plasmid display system allows the rapid and facile in vitro selection and can be a useful tool in discovering functional proteins from large libraries.

In vitro selection and identifcation of a cold‑tolerant variant in pineapple (Ananas comosus)

Yafen Zhang,Zhiqiang Xu,Tao Xie,Wei Zhang,Yehua He,Chaoyang Liu 한국원예학회 2022 Horticulture, Environment, and Biotechnology Vol.63 No.2

Cold stress is an important factor limiting the growth and distribution of pineapple. Breeding cold-resistant germplasm is an efective way to cope with this problem. In vitro selection for the somaclonal variation using diferent selection agents has been used for crop improvement in stress tolerance. In this study, the pineapple cold-tolerant variant was developed through in vitro cold selection of somaclonal variations. Low temperature was used as the selected agent, and the extreme lethal condition for the in vitro pineapple cultures was determined to be 0 °C for 72 h. The morphology changes of the in vitro cultures during the cold selection were observed and analyzed. The cold-tolerant variant line was fnally obtained through three consecutive selections with cold shock treatments, based on the established high-efciency culture system for pineapple embryogenic calli. The genetic variations at the molecular level in the cold-tolerant variant were verifed by ISSR analysis. The signifcantly improved cold tolerance in our selected variant was mainly refected by the higher survival rate, increased proline content, and elevated SOD activity under cold stress compared to these qualities in the control plants. This study demonstrated the feasibility of in vitro selection for cold tolerance in pineapple. The cold-tolerant variant could be valuable for future pineapple breeding programs and for cold tolerance research.

벼 꽃가루 캘러스의 저온처리에 의한 내연성 기내선발

양세준,오병근 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.1

내냉성의 지표로 이용되는 적고의 발현정도가 상이한 벼품종 및 유성계통과 그들이 상호교잡된 F$_1$씨 청배양에서 최초 형성된 캘러스(primary callus)를 저온 스트레스 (stress) 처리하여 내냉성 세포의 기내선발 가능성을 검토하였다. 캘러스 형성율이나 저온 스트레스를 통한 식물체 분화율은 자포니카형이나 자포니카형/통일형 조합이 통일형이나 통일형간 조합보다 높았으며 저온 스트레스 처리일수의 증가에 따라 백색체 분화율이 증가하는 경향을 보였다. 약배양 효율에 있어서 상호교잡에 따른 세질효과는 나타나지 많았다. 고정도가 높은 품종이나 교잡 F$_1$의 약배양 후대 계통에서 캘러스의 저온 stress처리일수에 따른 적고 발현정도의 변이가 인정되지 많았다. 적고발현 정도가 상이한 품종간 교잡 F$_1$에서 유래된 연배양 후대 계통의 적고 발현은 양친 범위 내에 대부분이 분포하였으나 적고 발현이 낮은 방향의 초월분리계통도 나타났다. 최초 형성된 캘러스의 저온 stress 처리($0^{\circ}C$ 10일)에서 분화된 약배양 계통, 무처리에서 분화된 약배양 계통과 F$_2$ 집단간에는 적고 배양의 빈도 분포에서 차이가 인정되지 않았다. 따라서 벼 약배양에서 최초 형성된 캘러스 수준에서 저온 스트레스를 통해 내냉성 세포를 직접적인 기내선발방법은 효과적이지 못함이 밝혀졌다. Is obtain cell lines showing high level of rice cold tolerance, direct in vitro selection through cold stress on primary pollen callus derived from anther culture was carried out Genotypic difference in callus formation and plant regeneration was recognized Rates of albino was increased along the duration of cold stress. Reciprocal effects were not noticed in anther culturability There was no variants related to rice leaf discoloration in pollen derived lines from parental varieties, regardless of days of cold stress. The regeneration and recombination of rice leaf discoloration in 146 pollen-derived lines, 70 pollen-derived lines from cold stress at $0^{\circ}C$ for 10 days, and 830 F$_2$ plants presented normal distribution curves with skewness in tolerance and no significant difference among 3 populations. Direct in vitro selection for rice cold tolerance through cold stress on primary pollen callus derived from anther culture, therefore, was revealed ineffective as a in vitro technology.

Evaluation of in vitro Water Stress Tolerance among EMS - Induced Variants of Banana (Musa spp., AAA), Using "Morphological, Physiological and Molecular" Traits

Bidabadi, Siamak Shirani,Mahmood, Maziah,Meon, Sariah,Wahab, Zakaria,Ghobadi, Cyrus 한국작물학회 2011 Journal of crop science and biotechnology Vol.14 No.4

Water stress is a serious environmental restriction to banana productivity. Hence, the objective of this study was to employ in vitro mutagenesis in selection and characterization of drought tolerant lines in banana. In vitro culture responses of ethyl methanesulphonate induced variants of banana cultivars, 'Berangan Intan' and 'Berangan' were assessed concerning morphological, physiological and molecular characteristics involving mutated shoot tips on MS medium supplemented with 30 g $L^{-1}$ PEG. The results showed that water stress tolerant lines could be obtained from induced variations. Variants $L_{2-5}$ and $L_{1-5}$ showed the highest number of leaves per shoot (2.37 and 2.06, respectively) and the lowest were recorded in the parental lines $L_{1-1}$ and $L_{2-1}$ (0.81 and 0.93, respectively). Fresh weight and shoot vigor rate indicated the maximum increase in the water stress tolerant lines compared with susceptible and non-mutated parental lines. $L_{2-5}$ exhibited the most increase in the chlorophyll and the most reduction in $H_2O_2$ and MDA contents when exposed to water stress. Under PEG treatment, proline and relative water content was enhanced in $L_{1-5}$, $L_{2-5}$, $L_{2-6}$, $L_{1-6}$, $L_{2-3}$, $L_{2-4}$, and $L_{1-4}$. RAPD analysis revealed polymorphism (18.35 and 21.48%) among variants derived from 'Berangan Intan' and 'Berangan', respectively. The amplified fragments generated by primers opc01, opc04, opa11, and opa20 observed to be specific for $L_{2-5}$ and $L_{1-5}$ as more tolerant followed by $L_{2-3}$, $L_{1-4}$, $L_{2-6}$, and $L_{1-6}$ as moderately tolerant lines against water stress. This study demonstrates the application of in vitro mutagenesis in selection of water stress tolerant lines of banana as a convenient, cheap, and rapid technique.