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Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

ABSTRACT: Presented here is an antisense-oriented method for functional gene screening, which we propose naming 'antisense display'. In principle, it consists of four steps: (i) preparation of phosphorothioate antisense repertoires that would correspond to the Kozak's consensus sequence, (ii) subgroup screening to identify active antisense molecules that could cause changes in the cellular phenotypes concerned and (iii) RT-PCR cloning of cDNA with the 5[prime] sense complement and 3[prime] anchor primers and sequence determination, followed by (iv) functional assays of candidate genes. Cell-free translation in rabbit reticulocyte lysate revealed that 10mer or longer antisense effectively halted protein synthesis. This required the presence of RNase H, and was achieved without prior heat-denaturation of the RNA templates. Then, subpools of the 10mer repertoire were administered to human microvascular endothelial cells in culture, and screened for anti-angiogenic activities. A single species having the sequence 5[prime]-GGCTCATGGT-3[prime] consistently inhibited the endothelial cell growth under hypoxia. Through RT-PCR with the corresponding sense primer, we came across three candidate cDNAs. Experiments employing longer unique antisense reproduced marked growth inhibitions in two of the three cDNAs. One encoded a mitochondrial protein and the other, which encoded a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand like domains, was a gene previously undescribed in human. The results suggest that the antisense display method is potentially useful for isolating new genes towards elucidating their functions.

SUBMITTER: Yonekura H

PROVIDER: S-EPMC148466 | biostudies-other | 1999 Jul

REPOSITORIES: biostudies-other

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Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

Yonekura H H Migita H H Sakurai S S Wang H H Harada S S Abedin M J MJ Yamagishi S S Yamamoto H H

Nucleic acids research 19990701 13

Presented here is an antisense-oriented method for functional gene screening, which we propose naming 'antisense display'. In principle, it consists of four steps: (i) preparation of phosphorothioate antisense repertoires that would correspond to the Kozak's consensus sequence, (ii) subgroup screening to identify active antisense molecules that could cause changes in the cellular phenotypes concerned and (iii) RT-PCR cloning of cDNA with the 5[prime] sense complement and 3[prime] anchor primers ...[more]

PMID: 10373574

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Project description:Background The use of head mounted display (HMD)-based immersive virtual reality (VR) coaching systems (HMD-VRC) is expected to be effective for skill acquisition in radiography. The usefulness of HMD-VRC has been reported in many previous studies. However, previous studies have evaluated the effectiveness of HMD-VRC only through questionnaires. HMD-VRC has difficulties in palpation and patient interaction compared to real-world training. It is expected that these issues will have an impact on proficiency. The purpose of this study is to determine the impact of VR constraints in HMD-VRC, especially palpation and patient interaction, on radiographic skills proficiency in a real-world setting. Methods First-year students (n = 30) at a training school for radiology technologists in Japan were randomly divided into two groups, one using HMD-VRC (HMD-VRC group) and the other practicing with conventional physical equipment (RP group) and trained for approximately one hour. The teachers then evaluated the students for proficiency using a rubric method. Results In this study, it was found that some skills in the HMD-VRC group were equivalent to those of the RP group and some were significantly lower than those of the RP group. There was a significant decrease in proficiency in skills related to palpation and patient interaction. Conclusions This study suggests that HMD-VRC can be less effective than real-world training in radiographic techniques, which require palpation and patient interaction. For effective training, it is important to objectively evaluate proficiency in the real world, even for HMD-VRC with new technologies, such as haptic presentation and VR patient interaction. Trial registration The study was conducted with the approval of the Ethics Committee of International University of Health and Welfare (Approval No.21-Im-035, Registration date: September 28, 2021). Supplementary Information The online version contains supplementary material available at 10.1186/s12909-022-03645-8.

Dataset Information

Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

Publications

Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

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