Abstract
Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.
| Original language | English (US) |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 145-157 |
| Number of pages | 13 |
| DOIs | |
| State | Published - Jan 1 2019 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1907 |
| ISSN (Print) | 1064-3745 |
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Keywords
- Cell-based screens
- DNA transposons
- Ex vivo forward genetic screening
- Functional annotation
- Growth-factor independence
- Sleeping Beauty
- Transposon mutagenesis
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery. / O'Donnell-Mendell, Kathryn A; Guo, Yabin; Suresh, Shruthy; Updegraff, Barrett L.; Zhou, Xiaorong.
Methods in Molecular Biology. Humana Press Inc., 2019. p. 145-157 (Methods in Molecular Biology; Vol. 1907).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery
AU - O'Donnell-Mendell, Kathryn A
AU - Guo, Yabin
AU - Suresh, Shruthy
AU - Updegraff, Barrett L.
AU - Zhou, Xiaorong
PY - 2019/1/1
Y1 - 2019/1/1
N2 - Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.
AB - Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.
KW - Cell-based screens
KW - DNA transposons
KW - Ex vivo forward genetic screening
KW - Functional annotation
KW - Growth-factor independence
KW - Sleeping Beauty
KW - Transposon mutagenesis
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UR - http://www.scopus.com/inward/citedby.url?scp=85058611131&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-8967-6_12
DO - 10.1007/978-1-4939-8967-6_12
M3 - Chapter
T3 - Methods in Molecular Biology
SP - 145
EP - 157
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -