GSBS Student Publications

Title

Generation of high-titer RCAS virus from DF1 chicken fibroblasts

Student Author(s)

Leanne G. Ahronian

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

Link to Article in PubMed

Journal Title

Cold Spring Harbor protocols

PubMed ID

25368307