Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

Abstract

We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation.

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

“…The inclusion of ARE element in the poly(A)-trap vector markedly improved the enrichment of authentic poly(A)-trapping events by reduction of the SD read-through background. These data suggest an advantage of our ARE-containing poly(A)-trap vector over the non-ARE-containing one and other poly(A)-trap vectors with a reverse insertion preference (Chen et al, 2004). Furthermore, trap inserts appeared to randomly integrate into targeted genes since insertions generated by our vector did not exhibit the severe 3 0 -bias observed with the widely used poly(A)-trap vectors (Ruley et al, 2005;Shigeoka et al, 2005).…”

“…To circumvent this problem, a number of expressionindependent or poly(A)-trap vectors have been developed and successfully utilized for the identification of differentially expressed genes (Chen et al, 2004;Lin et al, 2006;Tsakiridis et al, 2009). Basic poly(A)-trap vector contains are porter/ selectable marker gene flanked by an upstream constitutive promoter and a downstream splice donor (SD).…”

“…Although there are several versions of poly(A)-trapping vectors that are used in various vertebrate systems (Chen et al, 2004;Lin et al, 2006;Tsakiridis et al, 2009), there is room for improvement to avoid potential 3 0 -bias of integration and SD read-through. Accordingly, we generated a genefinding cassette by insertion of a modified IRES at the eleventh ATG site downstream of the reporter gene and an ARE element into the intron of SD signal.…”

Sphingolipids and membrane biology as determined from genetic models

“…In addition, Dictyostelium discoideum S1P lyase mutants are resistant to cis-platin treatment, have defects in their ability to form migrating slugs, and show defects in F-actin distribution, again linking sphingolipid metabolism with membrane dynamics [76,77]. S1P lyase (called SGPL in mouse) has been identified as an immediate early gene (IEG) product of PDGF activation [78]. In a recent report on mouse knock-out lines for twelve of these IEGs, including SGPL, all the lines showed similar phenotypes, in spite of the disparate functions ascribed to the individual proteins.…”

Oncogenic Serine-Threonine Kinase Receptor-Associated Protein Modulates the Function of Ewing Sarcoma Protein through a Novel Mechanism

“…A recent study suggests that STRAP may be a predictive marker of 5-fluorouracil (5-FU)-based adjuvant chemotherapy benefit in colorectal cancer (7). The homozygous STRAP null allele embryos showed recessive embryonic lethality between embryonic days E 10.5 and E 12.5 and had defects in angiogenesis, cardiogenesis, somatogenesis, neural tube closure, and embryonic turning (20). These results suggest a broader role of STRAP in tumorigenesis and development.…”