Identification of insect cell lines and cell line cross-contamination (CLCC) by internal transcribed spacer region of ribosomal DNA
Date Issued
2008
Date
2008
Author(s)
Lin, Hsueh-Fang
Abstract
Insect cell lines are useful tools in the study of virus basic biology and production of recombinant proteins, especially by baculovirus expression vector system (BEVS). In most cell culture laboratories, several different cell lines are routinely used, maintained, or handled, these cell lines should be regularly monitored to avoid cell line cross-contamination (CLCC). We developed a technique for identification of species origin and CLCC detection for insect cell lines (NTU-LY, NTU-PN, IPLB-LD652Y, and Sf9 cell lines) based on the sequence of internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The identity of the amplicon’s sequences between insect and its homologous cell line was up to 95 %~99 %, thus these amplicon’s sequences could be used to confirm the species origin of a cell line. Two endonucleases, PstI and HindII based on these sequences of amplicons, were selected for rapid identification of insect cell lines by PCR-RFLP (PCR-restriction fragment length polymorphism). The homologous cell lines could be examined by RAPD-PCR (random amplified polymorphic DNA – PCR) method with a selected oligonucleotide primer, OPU-10. Three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1, and Sf9-F2/ITS4, were then designated for identification of LY, LD, and Sf9 cells, respectively, by SS-PCR (species-specific PCR). Therefore three methods, RFLP-PCR, RAPD and SS-PCR, can be used to define cell origin and avoid CLCC. Otherwise, the mass production of NTU-LY cells was also undertaken by suspension culture after confirmations of cell origin and free from CLCC. The optimal condition of LY cell suspension culture was 1 x 106 cells/ml, in 200 ml culture volume and 65 RPM at 28℃. The LyxyNPV OBs (occlusion bodies) production reached to 70.5 ~ 97 OBs/cell that was much higher than monolayer infection (27.7 OBs/cell).
Subjects
insect cell lines
internal transcribed spacer (ITS)
ribosomal DNA (rDNA)
cell line cross-contamination (CLCC)
suspension culture
Type
thesis
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