Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 99, Issue 13, Pages 8838-8843Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.132274799
Keywords
-
Categories
Funding
- NCI NIH HHS [R01 CA077314, R01 CA077314-04] Funding Source: Medline
- NIAID NIH HHS [AI42252] Funding Source: Medline
Ask authors/readers for more resources
Genes regulating responses in mammalian cells are often difficult to identify by functional cloning strategies limited to a single round of selection. Here we describe a strategy, cyclical packaging rescue (CPR), which allows rapid recovery and retransmission of retroviral cDNA libraries. CPR can be used not only with immortalized cell lines such as fibroblasts and Jurkat T cells, but also with primary B lymphocytes, which can be maintained only in short-term cultures. CPR allows for multiple rounds of selection and enrichment to identify cDNAs regulating responses in mammalian cells. Using CPR, five cDNAs were functionally cloned, which conferred protection against tumor necrosis factor alpha (TNFalpha)-induced apoptosis in RelA(-/-) fibroblasts. Three of the genes, RelA, cellular FLICE-like inhibitory protein (c-FLIP), and a dominant-negative mutant of TNF receptor 1 arising through CPR afforded strong protection against apoptosis. Two of the genes identified, Dbs and Fas-associated death domain protein (FADD), previously identified as a proapoptotic molecule, afforded partial protection against TNFalpha-induced apoptosis. These results suggest that CPR is a versatile method that permits functional identification of both wild-type and dominant-negative gene products that regulate cellular responses.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available