Development of Cell-Defined Lentivirus-Based Microarray for Mammalian Cells

Author:

Kim Hi Chul12,Shum David1,Seol Hyang Sook3,Jang Se Jin3,Cho Ssang-Goo2,Kwon Yong-Jun14

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

3. Asan Institute for Life Science, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea

4. Ksilink, 16, Rue d’Ankara 67000 Strasbourg, France

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 × 108 infectious 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.

Publisher

Elsevier BV

Subject

Molecular Medicine,Biochemistry,Analytical Chemistry,Biotechnology

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