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Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System.

ABSTRACT: Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 10⁶ seeded HeLaS3 cells. 5' RACE analyses identified the well-known CYP24A1 gene as a calcitriol-responsive gene, as well as two new bexarotene- or calcitriol-responsive genes, BDKRB2 and TSKU, respectively. TSKU, interestingly, also responded to bexarotene. Endogenous levels of the TSKU and BDKRB2 transcripts displayed only slight changes and were not detected in the comprehensive analyses performed to date. Dose-response analyses of BDKRB2 and TSKU reporter cells in parallel revealed a differential profile in response to each vitamin A agonist, suggesting a bioanalyzer. The present study demonstrates that producing multiple reporter cells by a type of random screening can efficiently identify novel genes with unusual characteristics and be used for the profiling of the properties of vitamin compounds. Similar approaches to the method shown here may be useful for identifying new markers and for the analysis or diagnosis of nutrients, toxins, metabolites, etc.

SUBMITTER: Ishikawa K

PROVIDER: S-EPMC9409033 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System.

Ishikawa Kosuke K Tamamura Sakura S Takahashi Nobuhito N Takagi Motoki M Semba Kentaro K Watanabe Shinya S

International journal of molecular sciences 20220819 16

Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 106 seeded HeLaS3 cells. 5' RACE analyses identified the well-known CYP24A1< ...[more]

PMID: 36012634

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Project description:BackgroundLivestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance.ResultsIn this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production.ConclusionThese data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.

Dataset Information

Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System.

Publications

Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a <i>piggyBac</i> Transposon Promoter-Trapping Vector System.

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