Abstract
Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the in situ detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence in situ hybridization (sm-FISH), in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing's sarcoma cells. This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts.
Highlights
Chromosomal aberrations, including deletions, inversions, and translocations, which occur as the result of genotoxic stress, sometimes lead to the development of cancer [1,2]
Design of Fusion fluorescence in situ hybridization (FISH) In order to detect single molecules of fusion transcripts, we utilized single-molecule FISH probes, which were invented in our laboratory [15]
The simultaneous binding of so many probes to each target mRNA molecule causes the target mRNA molecules to each appear as a diffraction-limited spot in a fluorescence microscope [15]
Summary
Chromosomal aberrations, including deletions, inversions, and translocations, which occur as the result of genotoxic stress, sometimes lead to the development of cancer [1,2]. The most common type of chromosomal aberration associated with cancer development is chromosomal translocation, which involves the rearrangement of portions of nonhomologous chromosomes, leading to the fusion of two otherwise separate genes [3]. The products of these gene fusions can cause the abnormal expression of transcription factors or the unregulated activation of tyrosine kinases, both of which lead to uncontrolled cell growth, causing cancer [4]. Gene fusions were found to be responsible for soft-tissue sarcomas, prostrate cancer, lung cancer, and certain solid tumors [3,4,6,7]. With the advent of genome-wide translocation sequencing techniques, there is a growing list of tumors possessing an underlying gene fusion [7,8,9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
