Avian Retrovirus‐Mediated Tumor‐Specific Gene Knockout

Wei Wang1, Bingning Dong1, Feng Yang1

1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.17
DOI:  10.1002/cpmb.54
Online Posting Date:  January, 2018
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Abstract

The RCAS (replication‐competent avian sarcoma leukosis virus long‐terminal repeat with splice acceptor)‐TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI‐Oncogene (RCAS‐Cre‐IRES‐Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis. This versatile tumor gene modification system can also be adapted into different configurations to address different questions in appropriate mutant mouse hosts. Here we describe a protocol using the TuKO approach to knock out a gene of interest in tumors in appropriate hosts. © 2018 by John Wiley & Sons, Inc.

Keywords: Cre; RCAS; RCI; RCI‐PyMT; RCI‐Oncogene; TVA; tumor specific knockout

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Packaging, Collection, and Concentration of the RCI‐Oncogene Viruses
  • Basic Protocol 2: Titration of the RCI‐Oncogene Viruses
  • Basic Protocol 3: Injection of the RCI‐Oncogene Viruses into the Appropriate Host Mice for Tumor‐Specific Gene Knockout
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Packaging, Collection, and Concentration of the RCI‐Oncogene Viruses

  Materials
  • DF1‐Z/EG cells (see Wang et al., )
  • High‐glucose DMEM (e.g., HyClone, cat. no. SH30022)
  • FBS (e.g., Hyclone, cat. no. SH30071)
  • G418 disulfate salt (e.g., Sigma, cat. no. A1720)
  • RCI‐PyMT vector (see Wang et al., )
  • LipoD293 transfection reagent (e.g., SignaGen, cat. no. SL100668)
  • 60‐mm, 100‐mm, and 150‐mm cell culture dishes
  • 50‐ml conical tubes
  • Ultracentrifuge tubes (e.g., Beckman 38.5‐ml thinwall polypropylene tubes, cat. no. 326823)
  • Beckman XL‐80 ultracentrifuge, or equivalent
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Titration of the RCI‐Oncogene Viruses

  Materials
  • DF1‐Z/EG cells (see Wang et al., ).
  • DMEM (e.g., HyClone, cat. no. SH30022)
  • FBS (e.g., Hyclone, cat. no. SH30071)
  • G418 disulfate salt (e.g., Sigma, cat. no. A1720)
  • RCI‐Oncogene (see protocol 1)
  • 24‐well plates
  • Fluorescent microscope

Basic Protocol 3: Injection of the RCI‐Oncogene Viruses into the Appropriate Host Mice for Tumor‐Specific Gene Knockout

  Materials
  • fgfr1loxP/loxP mice on FVB background (see Pirvola et al., ; Yang et al., )
  • K19‐tva mice (provided by Dr. Brian Lewis; see also Morton et al., )
  • RCI‐Oncogene virus (see Basic Protocols protocol 11 and protocol 22)
  • Injectable anesthetic for mice
  • Bromophenol blue sodium salt (e.g., Sigma, cat. no. B5525)
  • Surgical scissors (e.g., SuperFine Vannas Scissors, curved, or equivalent)
  • 22‐G blunt‐tip needle
  • 50‐µl syringe (e.g., Hamilton, cat. no. 1705)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Figures

Videos

Literature Cited

Literature Cited
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  Wang, W., Meng, Y., Dong, B., Dong, J., Ittmann, M. M., Creighton, C. J., … Yang, F. (2017). A versatile tumor gene deletion system reveals a crucial role for FGFR1 in breast cancer metastasis. Neoplasia, 19, 421–428. doi: 10.1016/j.neo.2017.03.003.
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