测定水泡性口炎病毒中的基因强性

Isabel  S. Novella

doi:10.21769/BioProtoc.1073

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Measuring Genetic Robustness in Vesicular Stomatitis Virus

测定水泡性口炎病毒中的基因强性

Isabel S. Novella email

发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1073 浏览次数: 8799

评审: Anonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

May 2013

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

从大气颗粒物中分离细菌用于流式细胞计数的步骤

Carolina M. Araya [...] Isabel Reche

2019年06月20日 4888 阅读

水稻条纹病毒纯化

Gang Lu [...] Xiaorong Tao

2020年03月20日 3675 阅读

基于转化相关重组的酵母体系组装与诱变人冠状病毒OC43基因组

Brett A. Duguay and Craig McCormick

2025年08月20日 1006 阅读

Abstract

Genetic robustness is the ability of a genome to incorporate mutations with the result of no fitness changes. Thus, more robust viruses have an increased neutral mutation rate. This property is particularly important in RNA viruses due to their high mutation rates. The most direct way of measuring robustness in vesicular stomatitis virus (VSV) is to carry out clonal analysis of populations: randomly isolating individual VSV strains (plaques), measuring the fitness of each one and generating fitness distributions (Novella et al., 2010). A second possibility is to carry out multiple replicates of repeated plaque-to-plaque passages, determining fitness in progeny populations and generating fitness distributions (Novella et al., 2010). Depending on the expected differences, the former may require hundreds of determinations, while the latter may require tens of determinations. A third approach consists of increasing the mutation rate of populations under analysis to magnify any differences that may exist and, instead of measuring fitness, measuring survival (Novella et al., 2013). One caveat of this method is that changes in survival can also be explained by changes in polymerase fidelity. For that reason, it is important to perform complementary experiments, in this case quantifying mutant frequency.

Materials and Reagents

Test and reference VSV strains
Note : The former is the strain under investigation, the latter is the control (typically the progenitor).
Baby hamster kidney cells (BHK-21)
10x Trypsin/EDTA (Life Technologies, Gibco^®, catalog number: 15400 )
I1 Monoclonal antibody (I1Mab) hybridoma (Holland et al., 1991) (ATCC, catalog number: CRL-2700 )
Note: This antibody recognizes the G glycoprotein of VSV.
I14 Mab (Holland et al., 1991) (https://www.kerafast.com/p-172-hybridoma-ie9f9-i14.aspx)
Note: This antibody recognizes the G glycoprotein of VSV.
Proteose peptone No. 3 (PP3) (BD Difco^TM, catalog number: 212230 ) (12 g/L in dH₂O, autoclaved)
Bovine Calf Serum (BCS) (Life Technologies, Gibco^®, catalog number: 16170-078 )
Note: BCS is a good choice to carry out plaque assays and it is a lot cheaper than FBS.
Fetal Bovine Serum (FBS) (Life Technologies, Gibco^®, catalog number: 10437 )
10% CO₂
5-Fluorouracyl (5-FU) (Sigma-Aldrich, catalog number: F-6627 ) (10 mg/ml in ethanol, filtered)
Agarose (Lonza, Seqplaque^TM GTG^TM, catalog number: 50111 ) (40 g/L in dH₂O, autoclaved)
4.2% bicarbonate
Penicillin/streptomycin mixture (Mediatech, Cellgro^®, catalog number: 30-004-Cl )
Saline solution (see Recipes)
Minimal Essential Medium with Hank’s salts (MEM-H) (Mediatech, Cellgro^®, catalog number: 50-019-PB ) (see Recipes)
Crystal violet solution (see Recipes)