GSBS Student Publications
Title
Generation of high-titer RCAS virus from DF1 chicken fibroblasts
GSBS Program
Cancer Biology
UMMS Affiliation
Program in Gene Function and Expression; Department of Cancer Biology
Date
11-3-2014
Document Type
Article
Medical Subject Headings
Animals; Avian Sarcoma Viruses; Cell Line; Chickens; Fibroblasts; Genetic Vectors; Temperature; Virus Cultivation
Disciplines
Cancer Biology | Genetics and Genomics | Laboratory and Basic Science Research | Virology
Abstract
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.
Rights and Permissions
Citation: Cold Spring Harb Protoc. 2014 Nov 3;2014(11):1161-6. doi: 10.1101/pdb.prot077974. Link to article on publisher's site
DOI of Published Version
10.1101/pdb.prot077974
Related Resources
Journal Title
Cold Spring Harbor protocols
PubMed ID
25368307
Repository Citation
Ahronian, Leanne G. and Lewis, Brian C., "Generation of high-titer RCAS virus from DF1 chicken fibroblasts" (2014). GSBS Student Publications. 1918.
https://escholarship.umassmed.edu/gsbs_sp/1918