Title

Generation of high-titer RCAS virus from DF1 chicken fibroblasts

Student Author(s)

Leanne G. Ahronian

Academic Program

Cancer Biology

UMMS Affiliation

Program in Gene Function and Expression; Department of Cancer Biology

Publication Date

2014-11-03

Document Type

Article

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.

DOI of Published Version

10.1101/pdb.prot077974

Source

Cold Spring Harb Protoc. 2014 Nov 3;2014(11):1161-6. doi: 10.1101/pdb.prot077974. Link to article on publisher's site

Journal/Book/Conference Title

Cold Spring Harbor protocols

Related Resources

Link to Article in PubMed

PubMed ID

25368307

Link to Full Text

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