TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

The present study addressed whether retroviral vectors could be modified to achieve receptor-mediated, dose-controlled, and transient delivery of proteins or nucleic acids into targeted cells. As a paradigm, we generated mouse leukemia virus-based vectors encoding the site-specific recombinase Cre. The vectors were disabled in primer binding site function, blocking reverse transcription of the virion mRNA. While reducing transgene insertion more than 1000-fold and abolishing toxic effects of constitutive Cre expression, transient Cre delivery was still highly efficient, receptor restricted, and insensitive to pharmacologic inhibition of reverse transcription. This form of Cre transfer required the retroviral packaging signal, cap-proximal positioning of the translation unit, as well as gag and env expression in producer cells, revealing retroviral mRNA transfer as the underlying mechanism. Thus, retrovirally delivered mRNA may serve as an immediate translation template if not being reverse transcribed. This approach allows multiple modifications for targeted and reversible cell manipulation with nucleic acids.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5 0 untranslated region (5 0 UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3 0 UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3 0 long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (C). The latter concept is new and allows the use of an intron in the 5 0 UTR, taking advantage of retroviral splice sites surrounding C. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10 6 infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).