Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone

2型登革病毒克隆的随机插入诱变

Jeffrey W. Perry Jeffrey W. Perry
AT Andrew W. Tai

发布: 2018年08月20日第8卷第16期 DOI: 10.21769/BioProtoc.2975 浏览次数: 6771

评审: Modesto Redrejo-RodriguezSesha Lakshmi Arathi PaluriAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
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.

Mar 2018

Advertisement

“科研可重复性与创新”系列网络研讨会
Technologies for RNA Detection
Special Issue on Protocols for Organoid Research
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

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

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 3450 阅读

利用规模化饲养飞虱在田间条件下实现 Fijivirus 的可控传播方法

利用规模化饲养飞虱在田间条件下实现 Fijivirus 的可控传播方法

Analía D. Dumón [...] María F. Mattio

2026年03月20日 267 阅读

一种用于单细胞水平定量寨卡病毒RNA的新型测序方法

一种用于单细胞水平定量寨卡病毒RNA的新型测序方法

Min Hao [...] Yang Zhou

2026年03月20日 434 阅读

Abstract

Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis. A library of viral genomes, each containing a single randomly placed small insertion, is selected by passaging in cell culture and the insertion sites can be identified using Next Generation Sequencing (NGS). Here we describe a protocol for transposon mutagenesis of the 16681 strain of dengue virus, serotype 2. Mutant dengue virus libraries containing short randomly placed insertions are passaged through mammalian cells and insertions are mapped by NGS of the viable progeny. The protocol is divided into four stages: transposon mutagenesis of a dengue cDNA clone, viral genome transfection into permissive cells, isolation of viral progeny genomes, and sequencing library preparation.

Keywords: Transposon (转座子) search Mutagenesis (诱变) search RNA virus (RNA病毒) search Dengue (登革热) search Flavivirus (黄病毒属) search

Background

A key aspect of understanding viral pathogenesis is elucidating the viral proteins’ functions during infection. However, viral proteins, particularly those encoded by compact viral genomes, are often multifunctional and therefore more challenging to study, in part because they are often difficult to tag (epitope tags, fluorescent proteins, etc.) in the context of an infectious virus genome without compromising viral infection. One workaround is to express individually tagged proteins in cells, which may result in an incomplete picture of the viral protein’s function as the other viral proteins are not present. It also does not directly determine if the functional tag interferes with the tagged protein’s function(s) in viral infection. Another approach is the empiric tagging of viral proteins in the context of an infectious virus genome, which ensures viral viability but is an inefficient process that is difficult to scale.

Transposon mutagenesis can help dissect the functions of proteins under various experimental conditions and has been used at a whole genome scale to elucidate the role of various proteins during microbial infections. This approach has been successfully applied to a number of positive-strand RNA viruses (Arumugaswami et al., 2008; Beitzel et al., 2010; Teterina et al., 2011; Thorne et al., 2012; Remenyi et al., 2014; Eyre et al., 2017; Fulton et al., 2017). By coupling the transposon mutagenesis approach to Next Generation Sequence, a map of sites in the viral genome that tolerate insertions can be determined with unprecedented resolution. Once these sites are identified, functional tags can be introduced into these sites through site-directed mutagenesis. This protocol describes whole-genome transposon insertion mapping applied to the 16681 strain of serotype 2 dengue virus.

Materials and Reagents

  1. Pipette tips (Fisher Scientific, catalog numbers: 02-707-80 ; 02-707-167 )
  2. 6-well plate (Corning, catalog number: 3506 )
  3. 0.22 μm sterile filter (Corning, catalog number: 431219 )
  4. Cell scraper (Corning, catalog number: 3010 )
  5. 10-beta chemically competent E. coli (New England Biolabs, catalog number: C3019H ) or equivalent–genotype used Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC)
  6. Primers
    1. Fragment A (SacI/NarI) forward with T7 sequence
      GAAATTAATACGACTCACTATAAGTTGTTAGTCTACGTG
    2. Fragment A (SacI/NarI) reverse
      GTCATAGTGGCGCCTACCATAACCATCACTCTTCCC
    3. Fragment B (NarI/EcoRV) forward
      GGTTATGGTAGGCGCCACTATGACGGATGAC
    4. Fragment B (NarI/EcoRV) reverse
      CTGCTTCCTGATATCTCTGCCTGGTCTTCCC
    5. Fragment C (EcoRV/XbaI) forward
      GACCAGGCAGAGATATCAGGAAGCAGTCCAATCC
    6. Fragment C (EcoRV/XbaI) reverse
      AGAACCTGTTGATTCAACAGCAC
    7. Ion Torrent P1 adaptor top oligo
      CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
    8. Ion Torrent P1 adaptor bottom oligo
      <phos>ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT
    9. Modified Ion Torrent A adaptor top oligo
      <phos>GGCCGCCTGAGTCGGAGACACGCAGGGATGAGATGGTT
    10. Modified Ion Torrent A adaptor bottom oligo
      CGGACTCAGCCTCTGTGCGTCCCTACTCTACC
  7. SacI (New England Biolabs, catalog number: R3156 )
  8. NarI (New England Biolabs, catalog number: R0191 )
  9. EcoRV (New England Biolabs, catalog number: R3195 )
  10. XbaI (New England Biolabs, catalog number: R0145 )
  11. NotI (New England Biolabs, catalog number: R0189 )
  12. Mutation Generation System kit (Thermo Fisher Scientific, catalog number: F-701 )
  13. Carbenicillin (Fisher Scientific, catalog number: BP26481 )
  14. Kanamycin (Thermo Fisher Scientific, catalog number: 11815024 )
  15. Promega Wizard Maxiprep kit (Promega, catalog number: A7270 )
  16. Promega Gel Purification kit (Promega, catalog number: A9281 )
  17. m7g(5')ppp(5')A RNA Cap Structure analog (New England Biolabs, catalog number: S1405S )
  18. Agarose (Thermo Fisher Scientific, catalog number: 16500100 )
  19. Q5 polymerase (New England Biolabs, catalog number: M0493 )
  20. T7 MEGAscript in vitro RNA translation kit (Thermo Fisher Scientific, catalog number: AM1334 )
  21. ZYMO Quick-RNA Viral kit (ZYMO RESEARCH, catalog number: R1034 )
  22. Applied Biosciences High Capacity cDNA kit (Thermo Fisher Scientific, catalog number: 4368814 )
  23. Ion Library Taqman Quantitation kit (Thermo Fisher Scientific, catalog number: 4468802 )
  24. 10x TBE (Fisher Scientific, catalog number: BP1333 )
  25. T4 DNA ligase (New England Biolabs, catalog number: M0202 )
  26. T4 PNK (New England Biolabs, catalog number: M0201 )
  27. dNTPs (Thermo Fisher Scientific, catalog number: R1121 )
  28. AMPure XP Beads (Beckman Coulter, catalog number: A63881 )
  29. LB medium (Fisher Scientific, catalog number: BP1425-500 )
  30. PEG 8000 (Thermo Fisher Scientific, catalog number: BP233-100 )
  31. Vero cells (ATCC, catalog number: CCL-81 )
  32. TransMessenger transfection reagent (QIAGEN, catalog number: 301525 )
  33. Full-length dengue virus clone 16681 serotype 2 cDNA (in plasmid pD2/IC-30P-NBX, Huang et al., 2010).
    Note: This cDNA was a generous gift of Dr. Huang from the Centers for Disease Control.

Equipment

Note: No specific equipment is necessary and any model of the following should suffice.

  1. Balance
  2. Pipettes
  3. Heat block
  4. Incubator
  5. Thermal cycler
  6. Water bath
  7. Microcentrifuge
  8. Magnet for Ampure XP bead purification

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Perry, J. W. and Tai, A. W. (2018). Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone. Bio-protocol 8(16): e2975. DOI: 10.21769/BioProtoc.2975.

ris ris Download Citation in RIS Format

分类

微生物学 > 微生物-宿主相互作用 > 病毒

微生物学 > 微生物遗传学 > 诱/突变

分子生物学 > DNA > 诱/突变

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件