Gemcitabine has been a first-line chemotherapy agent for advanced pancreatic cancer, which is associated with one of the lowest 5 years survival rates among human cancers. Due to our lack of understanding of the genetic determinants of Gemcitabine sensitivity in pancreatic cancer, the therapeutic effectiveness of Gemcitabine chemotherapy is typically unpredictable. Using a genome-wide and piggyBac transposon-based genetic screening platform, we identified the PVT1 gene as a regulator of Gemcitabine sensitivity and showed that functional inactivation of the PVT1 gene led to enhanced Gemcitabine sensitivity in human pancreatic cancer ASPC-1 cells. The integration of the piggyBac transposon-based vector system into intron 3 of PVT1 was within a common site of oncogenic retroviral insertions and chromosomal translocations. PVT1 is a non-protein encoding gene; the genomic arrangement of PVT1 and its co-amplification with MYC have been implicated in the tumorigenesis of a variety of cancers. The molecular mechanism of PVT1 transcripts in gene regulation remains a puzzle. We demonstrated that overexpression of a full length PVT1 cDNA in the antisense orientation reconstituted enhanced sensitivity to Gemcitabine in naive ASPC-1 cells, whereas overexpression of a full length PVT1 cDNA in the sense orientation resulted in decreased sensitivity to Gemcitabine. Our results identified PVT-1 as a regulator of Gemcitabine sensitivity in pancreatic cancer cells and validated the genome-wide genetic screening approach for the identification of genetic determinants as well as potential biomarkers for the rational design of Gemcitabine chemotherapies for pancreatic cancer. (C) 2011 Published by Elsevier Inc.