| RRC ID |
61868
|
| Author |
Shindoh N, Yoda A, Yoda Y, Sullivan TJ, Weigert O, Lane AA, Kopp N, Bird L, Rodig SJ, Fox EA, Weinstock DM.
|
| Title |
Next-generation cDNA screening for oncogene and resistance phenotypes.
|
| Journal |
PLoS One
|
| Abstract |
There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.
|
| Volume |
7(11)
|
| Pages |
e49201
|
| Published |
2012-1-1
|
| DOI |
10.1371/journal.pone.0049201
|
| PII |
PONE-D-12-22412
|
| PMID |
23145123
|
| PMC |
PMC3492311
|
| MeSH |
Animals
Cell Line
Cell Proliferation
Drug Resistance, Neoplasm / genetics*
Erlotinib Hydrochloride
Gene Library*
Genes, erbB-2
Genetic Predisposition to Disease
Genetic Testing / methods*
Genetic Variation
Mice
Oncogenes*
Phenotype
Protein Isoforms
Quinazolines / pharmacology
|
| IF |
2.74
|
| Resource |
| Human and Animal Cells |
Ba/F3(RCB0805) |
