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

Insect cell cultures are important for the study of basic insect cytology (Fallon 2008); for biological research into insect immunity and signal mechanisms (Gillespie et al. 1997; Stanley 2006; Stanley and Miller 2006); for the in vitro propagation of insect pathogens (especially viruses and microsporidia) for pest control (Shih et al. 1996; Wang et al. 1996; Wu et al. 2002; Sudeep et al. 2005; Wu and Wang 2006; Yeh et al. 2007; Tsai et al. 2009); for the screening and testing of novel chemical insecticides (Smagghe et al. 2009); and for the construction of baculovirus expression vector systems (BEVS) to produce recombinant proteins for use as pharmaceuticals and human vaccines (Bornstein 2009; Cox and Hollister 2009). Currently, there are over 500 continuous cell lines that have been established from more than 100 insect species (Lynn 2001, 2007) and over 900 viruses have been propagated in lepidopteran cell lines (Lynn 1999, 2007). However, all cellculture systems are susceptible to the frequent and recurrent problem of cell-line cross-contamination (CLCC). This problem exists in many mammalian cell lines, where as many as 20% of the cell lines were found to be contaminated with HeLa cells (Stacey 2000; Buehring et al. 2004), whereas National Testing Services report that 17–36% of cell cultures in use contain a misidentified species or cell type (Nelson-Ress 1978; Nelson-Ress et al. 1981; Markovic and Markovic 1998). At the very least, CLCC can invalidate research results (Wenzel and Daniel 2005). Compared with mammalian cell lines, crosscontamination and/or mislabeling in insect cell lines is thought to be even more widespread (Green and Charney 1971; McIntosh et al. 1996). In addition to traditional protein-based methods such as isoenzymes (Brown and Knudson 1980, 1982; Nims et al. 1998) and immunologic procedures (Aldridge and Knudson 1980), there are also several methods based on DNA molecular markers that might be used to detect CLCC in insect cells. In the present study, we investigate the practicalities of using the nuclear ribosomal internal transcribed spacer (ITS) region to address this problem. In eukaryotes, the ITS region of 18S–28S nuclear