The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site

Abstract

The internal ribosome entry site (IRES) from the picornaterial neo sequence has a negative effect on expression. virus family has frequently been used to express multiple Analysis of the steady-state RNA levels isolated from genes from a polycistronic message in retroviral vectors.transfected packaging cells showed that the neo-containWhile examining factors affecting levels of gene expression ing retroviral vectors produced significantly lower levels of in IRES-containing retroviral vectors, it was found that RNA than those lacking this bacterial sequence, indicating retroviral vectors expressing the two genes linked by IRES, that neo interferes with expression of the neighboring gene the reporter gene and the selectable marker neo, produced at the level of RNA. Furthermore, the order of genes in the significantly lower levels of protein than those containing a IRES-neo-containing vectors appeared to be more reporter gene alone. This observation has been made with important than in the vector lacking the neo sequence. Our various cDNA sequences. However, when the neo was results suggest that neo has to be used in the retroviral replaced with a different cDNA, the level of gene vector with care, especially when a high level gene expression was increased, often to the level achieved with expression is needed. a vector expressing a single gene, suggesting that the bacKeywords: IRES; neo; retrovirus; gene therapy Retroviral vectors have been the vehicle of choice for many gene transfer experiments and a large number of clinical protocols still employ ex vivo approaches which necessarily involve the use of selectable marker genes to allow easy selection for the successfully transduced cells. 1,2 Of the various dominant selectable markers that are in use for mammalian cells, the neo gene has been the most extensively employed as a marker for retroviral vectors. 3,4 There are two general ways of expressing neo along with the gene of interest. In one type, an independent promoter, either internal or the LTR itself, is used, 5 while in the second type, the neo gene is expressed as part of a multicistronic message using an internal ribosome entry site (IRES). 6-10 The internal promoter-based designs were thought to suffer from the limitation of promoter occlusion where the transcription from one promoter suppresses transcription from another when the two promoters are arranged in the same orientations. 11-13 IRESbased designs avoid such problems by expressing multiple genes in one message from a single promoter.The IRES sequences utilized to date are derived mostly from members of the picornavirus family. They can be isolated from an original viral genome and linked to Correspondence: S Kim, IMBG, Korea Received 26 February 1998; accepted 5 June 1998 unrelated genes to produce polycistronic mRNAs. Indeed, IRES elements have been demonstrated to work in the context of retroviral vectors to express two or more genes. [6][7][8][9][10] Despite the wide use of neo and IRES in retroviral vector designs, factors affecting gen...

Variegation of retroviral vector gene expression in myeloid cells

“…31,32 On the other hand, sequences other than those present in the LTR can influence viral transcription. 33 Indeed, the pBH vectors contain sequence elements, such as the hygromycin resistance gene and a prokaryotic origin of DNA replication, 12 that may contribute to the negative reciprocal interactions that results in poor or absent gp91-gene expression.…”

Loss of T cell-mediated antitumor immunity after construct-specific downregulation of retrovirally encoded T-cell receptor expression in vivo

“…Numerous other mechanisms that could play a contributory role to our observed loss of gene expression have also been reported. For example, expression of the neomycin resistance gene has been linked to problems with gene expression 41–43. Although we cannot directly exclude any of these mechanisms as contributory, our results suggest either (1) a problem directly associated with the SRα promoter's ability to maintain gene expression in vivo on murine T cells or (2) a problem indirectly related to the SRα promoter gene position in the specific retroviral construct used in these studies.…”

Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome

“…Again, MT‐IDS gave the highest level of IDS activity, on average 2‐ and 4‐fold higher levels than MIN‐IDS and two other vectors, respectively (Figure 3B). These results suggested that even the same LTR could give varying results when placed under different genomic environments, consistent with our previously published data 24.…”