A novel functional gene selection method provides a systematic view of cell migration

Abstract

Cell migration is a central process that is essential for embryonic development, wound repair, inflammatory response, homeostasis and tumor metastasis. A method of genome-wide selection based on the gain-of-function has been devised to identify novel cell migration-promoting genes in cultured cells. After the introduction of the retroviral mouse brain cDNA library into NIH3T3 mouse fibroblast cells, migration-promoted cells were selected by a three-dimensional migration assay using cell culture inserts. After five rounds of enrichment, cDNAs were retrieved from the cells that passed the selection processes. Cell migration-promoting activity was confirmed by independent migration assays for the retrieved cDNAs. Multiple cell migration-promoting genes were successfully isolated by this method. The genes identified can be used to gain a systematic view of cell migration. The gain-of-function selection method described here can be combined with RNAi-mediated loss-of-function screen or selection to be a more powerful tool for the systems biology research of cell migration.

Figure 1

Schematic illustration of novel gain-of-function in vitro selection for the cell migration-promoting genes. (A) A retroviral cDNA library was generated by transient transfection of Phoenix Eco packaging cells with ViraPort^® XR Mouse Brain cDNA Library (Stratagene). Cell-free supernatants were harvested 2 days post-transfection and subsequently used to infect NIH3T3 fibroblast cells at multiplicity of infection (MOI) of 1. (B) Two days post-infection, cells were seeded (4 × 104 cells/well) onto the transwell culture inserts and incubated at 37°C for 5 h. Migrated cells were collected by trypsinization of the cells adhered to the lower face of the transwell culture inserts or the bottom of the plates, and re-seeded onto the transwell culture inserts. Cells were then allowed to migrate again for enrichment, which was repeated five times. (C) After the final round of enrichment, migrated cells were expanded into individual clones by limiting dilution. (D) The chromosomally integrated cDNAs were identified by PCR followed by sequencing. The cDNA segments were amplified by using the primers that are specific for regions flanking the multiple cloning site of the retroviral vector. (E) Finally, identified cDNA fragments were cloned into pcDNA3 and further analyzed.