Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression.

Abstract

Recombinant adenoviruses were constructed in which the viral ElA gene was deleted and the E1B promoter was replaced by the rat albumin, mouse p-major globin, or mouse immunoglobulin heavy-chain promoter.After infection of human or rat hepatoma cells, ElB-containing mRNAs could be detected only from the virus containing the albumin promoter. Conversely, only the immunoglobulin promoter was active in virus-infected myeloma cells. However, in hepatoma cells transcription from the albumin promoter in the virus was much less than that of the endogenous cellular albumin gene or of other viral genes. In primary mouse hepatocytes endogenous albumin gene transcription was high immediately after plating but declined within 24 h. Expression of the albumin promoter in the virus paralleled that of the cellular albumin gene. From these results it appears that cell-specific expression of albumin depends on the presence of tissue-specific trans-acting factors, but the presence of such factors does not suffice for a maximal rate of transcription, a conclusion that requires direct comparison within a differentiated cell of a newly introduced and preexisting active cell gene.Tissue-specific gene expression in vertebrates appears most often to result from regulation of transcriptional initiation (11,12,36,41,44). In our previous experiments on gene control in mouse hepatocytes and cultured mouse and rat hepatoma cells, we have noted two aspects of transcriptional control: specificity among cell types (12, 36) and a higher differential rate of transcription for liver-specific genes by adult hepatocytes on the one hand and fetal hepatocytes, hepatoma cells, and cultured adult cells on the other (6-8, 36).While cell-specific expression of genes introduced by transfection into cultured differentiated cell lines from the pancreas, lens, the P-lymphocytic series, and liver has been demonstrated, (5,13,15,16,19,34,37,42), the direct measurement of transcription rates following transfection is difficult to determine. Moreover, transfection experiments do not afford a comparison between the rate of transcription of endogenous and newly introduced genes because only a small fraction of the exposed cells actually take up and express the plasmid DNA. A further difficulty in transfection experiments is that all cell types are not equally receptive to uptake or the expression of newly introduced DNA, making comparisons between cell types very difficult.Finally, the gene expression assay in most reported transfection experiments relies on the steady-state amount of an mRNA or protein product, often the levels of bacterial chloramphenicol acetyltransferase (CAT) activity produced from a fusion of the regulatory region of the tissue-specific gene with the gene encoding the CAT protein. Such steadystate measurements are usually made only after prolonged incubation time (24 to 72 h). Use of this assay to study transcriptional control requires the assumption that both an unusual mRNA and protein product are equally stable in different cells under diff...

Tightly regulated gene expression in human hematopoietic stem cells after transduction with helper-dependent Ad5/35 vectors

“…Ad ITRs have promoter activity and the Ad packaging signal overlaps with the E1A enhancer [12,13]. The presence of these regulatory elements, that cannot be removed from the vector backbone, affects tissue specific expression [14,15], and also causes high background expression levels without induction when Tet-on or Tet-off expression systems are incorporated into first-generation [16] or HD Ad5 vectors [17]. We and others have shown that this problem can be addressed by incorporating insulators [18][19][20] and/or polyadenylation signals [21] into the vector genome.…”

Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region.

“…Cells were transfected by using the DEAEdextran method as described by Lopata et al (17). Recombinant adenoviral vectors were constructed as described (18). Details of the cloning steps involved in the construction of plasmid and viral vectors are available upon request.…”

“…DNA fragments beginning at various distances 5' to the cap site and ending at the HindIII site within the intron (203 nt 3' to exon 1 splice donor site; nt 288 in Fig. 2 were fused to a marker sequence, a portion of the adenovirus type 5 Elb gene lacking its own promoter (18). Specific transcription from such a recombinant template can be detected by using a labeled RNA probe complementary to Elb in a RNase T2 protection assay as diagramed in Fig.…”

“…The second, inhibitory arm of the transcription cycle is more readily detected in normal diploid human fibroblasts than in the aneuploid transformed HeLa cells used for transfection. Due to the inefficiency of transfection of these fibroblasts using naked DNA, the -800 nt ISG-54K:Elb plasmid was incorporated into an adenovirus vector (18). Such viral vectors efficiently infect many cell types and thus serve as a convenient vehicle for enhanced transfer of test DNA sequences into normal cells.…”

Serum and v-src increase the level of a CCAAT-binding factor required for transcription from a retroviral long terminal repeat.

“…For the run-on assay, we blotted l lag per slot of the HindIII-NcoI fragment of RSVNEO, containing the neomycin phosphotransferase-coding portion, and 5 ~g per slot each of pFos (the 1.0-kb PstI fragment of murine v-los cloned into pUC18; Curran et al 1982), pTub {1.7-kb chicken [3-tubulin cDNA cloned into the PstI site of pBR322; Cleveland et al 1980), p28S (4.8-kb SatI-EcoRI fragment of mouse 288 rRNA genomic DNA clone in pBR322; Tiemeier et al 1977), pUC18, and 6X174 DNA. The assay, itself, was performed as described in Friedman et al (1986).…”