Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus

A recombinant adenovirus (rAd) expressing Cre recombinase derived from bacteriophage P1 has already been extensively used for the conditional gene activation and inactivation strategies in mammalian systems. In this study, we generated AxCAFLP, a rAd expressing FLP recombinase derived from Saccharomyces cerevisiae and carried out quantitative comparisons with Cre-expressing rAd in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells. In the in vitro experiments, the relative recombination efficiency of FLP expressed in 293 cells infected with FLP-expressing rAd was approximately one-thirtieth that of Cre even at 30°C, the optimum temperature for FLP activity, and was approximately one-ninetieth at 37°C. Co-infection experiments in HeLa cells using a target rAd conditionally expressing LacZ under the control of FLP showed that an FLP-expressing rAd, infected at a multiplicity of infection (MOI) of 5, was able to activate the transgene in almost 100% of HeLa cells whereas the Cre-expressing rAd was sufficient at an MOI of 0.2. Since an MOI of 5 is ordinarily used in rAd experiments, these results showed that the FLP-expressing rAd is useful for gene activation strategies and is probably applicable to a sequential gene regulation system in combination with Cre-expressing rAd in mammalian cells.

A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.

Isolation of a differentially regulated splicing isoform of human NF-E2.

The transcription factor NF-E2 (nuclear factor erythroid 2), interacting via DNA motifs within regulatory regions of several hematopoietic genes, is thought to mediate the enhancer activity of the globin locus control regions. By screening a human fetal liver cDNA library with probes derived from mouse NF-E2, we have isolated a splicing variant of the NF-E2 gene (fNF-E2) that differs in the 5' untranslated region from the previously reported cDNA (aNF-E2). The fNF-E2 isoform is transcribed from an alternative promoter located in the 3' end of the first intron and joined by alternative splicing to the second and third exons, which are shared by both RNA isoforms. Although the two forms produce the same protein, they are expressed in different ratios during development. fNF-E2 is more abundant in the fetal liver and less abundant in the adult bone marrow compared to the previously described form. Their distribution apparently follows the differential expression of fetal and adult hemoglobins.