title: Development of Cell-Defined Lentivirus-Based Microarray for Mammalian Cells.
abstract: Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 108infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.
authors.affiliation: 1 Institut Pasteur Korea, IP-Korea, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.|2 Department of Animal Biotechnology and Incurable Disease Animal Model & Stem Cell Institute (IDASI), Konkuk University, Seoul, Republic of Korea.
authors.forename: Hi Chul
authors.initials: HC
authors.lastname: Kim
authors.affiliation: 1 Institut Pasteur Korea, IP-Korea, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.affiliation: 3 Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Hyang Sook
authors.initials: HS
authors.lastname: Seol
authors.affiliation: 3 Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Se Jin
authors.initials: SJ
authors.lastname: Jang
authors.affiliation: 2 Department of Animal Biotechnology and Incurable Disease Animal Model & Stem Cell Institute (IDASI), Konkuk University, Seoul, Republic of Korea.
authors.forename: Ssang-Goo
authors.initials: SG
authors.lastname: Cho
authors.affiliation: 1 Institut Pasteur Korea, IP-Korea, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.|Ksilink, 16, Rue d'Ankara 67000 Strasbourg, France.
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Kwon
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D008322:Mammals / Q000378:metabolism*; D046228:Microarray Analysis / Q000379:methods*; D011107:Polylysine / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D001672:Biocompatible Materials; D049452:Green Fluorescent Proteins; D011107:Polylysine
keywords: cDNA; cell-defined array; lentivirus; microarray; reverse infection