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. 2002 Jun 25;99(13):8838-43.
doi: 10.1073/pnas.132274799.

Identification of gene function by cyclical packaging rescue of retroviral cDNA libraries

Deepta Bhattacharya  1 , Eric C Logue, Sonia Bakkour, James DeGregori, William C Sha
Affiliations

Identification of gene function by cyclical packaging rescue of retroviral cDNA libraries

Deepta Bhattacharya et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Rapid recovery and controlled retransmission of retroviruses from multiple cell lineages by using CPR. (A) Scheme for packaging rescue of stably integrated helper-free retroviruses from cells. Retrovirally transduced cells are infected with Adgag-pol and Adenv and washed. After 24 h, retroviral supernatants are recovered and used to retransduce fresh cells. (B) Retroviruses can be rapidly recovered from fibroblasts, primary B cells, and Jurkat T cells and retransmitted efficiently to fresh cells. Retroviral supernatants recovered 24 h after infection with Adgag-pol and Adenv were used to transduce a fresh lot fibroblasts, which can be enriched for retroviral marker expression and used to retransduce primary B cells or Jurkat T cells expressing the mCAT1 receptor. Gray-shaded plots represent controls in which one or both of the adenoviruses were omitted. The efficiency of packaging rescue is represented as the starting number of transduced cells required to retrovirally infect 105 target cells. (C) Rare marker retroviruses can be maintained through one round of CPR in fibroblasts and primary B cells. For fibroblasts, MSCV-GFP-RelA-IRES-puro-transduced cells were mixed with MSCV-IRES-puro-infected cells at ratios of ≈1:100 or 1:1,000, retroviruses were rescued by using CPR, and transferred to a fresh lot of RelA−/− fibroblasts. GFP-expressing cells were quantified by flow cytometry after puromycin selection. For primary B cells, MSCV-Thy1.1-IRES-PLAP-transduced cells were mixed with MSCV-IRES-Thy1.1-infected cells at a ratio of 1:10,000 and were subjected to one round of CPR by using fibroblast intermediates followed by flow cytometric analysis of Thy1.1 and enzymatic detection of PLAP.
Figure 2
Figure 2
Multiple rounds of a functional assay can be used by using CPR to amplify and identify rare regulating responses of cDNAs. Multiple rounds of selection for cDNAs conferring resistance to TNFα-induced apoptosis can be performed with CPR. RelA−/− fibroblasts with an integrated retrovirus expressing a GFP-RelA fusion protein were initially seeded at a frequency of 1:156,000 with RelA−/− fibroblasts with a control retrovirus. CPR was performed and after four rounds of selection, cells transduced with the GFP-RelA retrovirus were enriched 50,000-fold to a frequency of 1:3.
Figure 3
Figure 3
Genes functionally identified by CPR include dominant-negative mutants generated during the process of CPR. (A) Genomic DNA from TNFα-sensitive and TNFα-resistant pools of cells was subjected to PCR using retroviral-specific primers. “Initial” represents unselected retroviral library-transduced cells before TNFα treatment. Pool 12 remained sensitive to TNFα treatment after four rounds of CPR. Pools 13, 15, and 17 are representative of pools that became resistant to TNFα treatment. (B) The enriched cDNA for TNFR1 is a dominant-negative mutant that was generated during CPR in round 1. The region mutated in the TNFR1* clone identified in pool 13 was amplified by PCR and digested with XmnI. The mutant TNFR1* cDNA is missing an XmnI restriction site found in the wild-type TNFR1 cDNA.
Figure 4
Figure 4
Genes functionally identified by CPR include the modifiers FADD and Dbs* that only partially protect against TNFα-induced apoptosis. Amplified PCR bands were recloned into retroviral vectors and tested for their ability to confer protection against TNFα-induced apoptosis in RelA−/− fibroblasts. The fold-survival advantage of virally transduced cells over nontransduced cells after a single round of TNFα treatment was calculated as: percentage of infected cells after TNFα treatment, percentage of uninfected cells before TNFα treatment, percentage of infected cells before TNFα treatment, and percentage of uninfected cells after TNFα treatment. SDs from a minimum of six independent experiments are shown and ranges of values obtained for fold-survival advantage are shown in parentheses.
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