Student Authors
Leanne G. AhronianDocument Type
Journal ArticlePublication Date
2014-11-03Keywords
Animals; Avian Sarcoma Viruses; Cell Line; Chickens; Fibroblasts; Genetic Vectors; Temperature; Virus CultivationCancer Biology
Genetics and Genomics
Laboratory and Basic Science Research
Virology
Metadata
Show full item recordAbstract
RCAS viruses are replication-competent in avian cells, but are replication-deficient in mammalian cells. Therefore, high-titer RCAS virus stocks can be generated only in avian cells. The chicken fibroblast cell line DF1 is well suited for this purpose. Successful infection of target mammalian cells, particularly in vivo, is dependent on the production of high titer viruses by DF1 cells. Moreover, consistency in viral titer helps to ensure uniformity in results produced following the use of independent lots of virus producer cells. Therefore, it is critical to determine the viral titer before initiating these experiments. Because several factors, including insert size and the effect of the inserted gene product on the viability of DF1 cells, influence viral titer, the production of high virus titers cannot be assumed. For RCASBP-A-based viruses, a titer of > 1 x 10(7) IU/mL is considered appropriate. Importantly, the virus reverse transcriptase is error prone; errors will accumulate in the virus produced over time. Therefore, virus producer cells should not be cultured for > 4-6 wk before being replaced with fresh producer cells. Low passage virus producer cells may be frozen and stored at -80 degrees C; thawed cells will not display a reduction in virus titer. Virus can be collected regularly, concentrated, and stored at -80 degrees C for long-term use; thawed viral stocks typically show a 10-fold decrease in titer.Source
Cold Spring Harb Protoc. 2014 Nov 3;2014(11):1161-6. doi: 10.1101/pdb.prot077974. Link to article on publisher's siteDOI
10.1101/pdb.prot077974Permanent Link to this Item
http://hdl.handle.net/20.500.14038/33392PubMed ID
25368307Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1101/pdb.prot077974