Related Experiment Video
Updated: Aug 16, 2026
Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells
Published on: August 25, 2021
Identification of functional genes during Fas-mediated apoptosis using a randomly fragmented cDNA library
M Hassan1, A Mirmohammadsadegh, D Selimovic
1Institute of Pathology, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.
Abstract:
We describe a general strategy for the identification of functional genes that, when downregulated, result in a selectable phenotype. This strategy is based on expression selection of cDNA fragments that counteract their cognate genes. A cDNA library containing random fragments expressed in human HepG2, A375 and CLS-354 cells was used to identify functional genes whose inhibition conferred resistance to Fas-induced apoptosis. Thirty-five clones were isolated, 28 of which were derived from unknown genes, that tagged 19 individual genes and 7 of which referred to known genes that tagged the apoptosis-related protein (APR)-1, -2 and indoleamine-pyrrole 2,3,-dioxygenase (IDO). The ability of APR-1-, -2- and IDO-derived antisense RNAs to induce resistance to Fas in HepG2, A375 and CLS-354 cells suggested that APR-1, -2 and IDO genes are involved in the machinery of Fas-mediated apoptosis. Our gene discovery strategy provides a generally applicable procedure to identify functional genes that interfere with apoptosis, and may therefore be clinically relevant for tumor therapy.
Insights
This study introduces a novel gene discovery strategy to identify genes that, when inhibited, confer resistance to apoptosis. This method successfully identified genes involved in Fas-mediated apoptosis, offering potential for cancer therapy.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Identifying genes that regulate apoptosis is crucial for understanding cell death pathways.
- Targeting apoptosis is a key strategy in cancer therapy.
Purpose of the Study:
- To develop a general strategy for identifying functional genes that, upon downregulation, yield a selectable phenotype.
- To discover genes involved in Fas-induced apoptosis using expression selection of counteracting cDNA fragments.
Main Methods:
- Construction and screening of a human cDNA library in HepG2, A375, and CLS-354 cells.
- Utilizing expression selection to identify cDNA fragments that inhibit cognate gene function.
- Assessing the impact of identified gene inhibition on resistance to Fas-induced apoptosis.
Main Results:
- Successfully isolated 35 clones, identifying 19 novel genes and 7 known genes.
- Identified apoptosis-related protein (APR)-1, -2, and indoleamine-pyrrole 2,3,-dioxygenase (IDO) as key players.
- Demonstrated that antisense RNAs derived from APR-1, -2, and IDO confer resistance to Fas-induced apoptosis.
Conclusions:
- The developed gene discovery strategy is broadly applicable for identifying genes interfering with apoptosis.
- APR-1, -2, and IDO are implicated in the Fas-mediated apoptosis pathway.
- This approach holds clinical relevance for developing novel tumor therapies targeting apoptosis.
More Related Videos
12:42Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
Published on: April 13, 2012
10:06Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017