Identification of insect cell lines and cell-line cross-contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR-RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR-based method used three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1 and Sf9-F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell-line cross-contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell-line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.