Variegation and silencing in a lentiviral-based murine transgenic model

Abstract

Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis and germ line transmission, a wide variation in transgene expression was observed in most first and second generation mouse lines. In particular, despite the segregation of integrants into single-copy expressing mouse lines, transgene expression appeared to be the target of epigenetic regulatory mechanism, often causing the coexistence of high and low transgene expressing cells within a given tissue such as blood peripheral lymphocytes. The establishment and analysis of large number of mouse lines may therefore be required to select a stable transgenic line with pancellular expression of a gene of interest using this lentiviral-based approach.

A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells

“…[9][10][11] However, despite widespread use of lentviralbased transgenics, significant challenges remain. [12][13][14][15][16][17] Chief among these challenges is achieving consistent, high-level expression and overcoming transgene silencing. To this end, expression of multiple genes from a single expression cassette is a desirable characteristic, as it enables selectable agents or marker genes to be co-expressed, 2 permits manageable construct size and achieves constant production of desired gene products.…”

“…27,29,36,37 Within this context, however, promoter choice is critical to successful experimental outcomes, especially in stem cells where transgene silencing is frequently observed. 13,37 Thus, to be feasible, these bidirectional promoters must possess strong transcriptional activity in a variety of cell types and resist epigenetic silencing commonly associated with stem cell biology and animal transgenics. 13,36,37,43 Here, our goal was to develop a bidirectional lentiviral vector exhibiting strong transcriptional activity with which to conduct RNA interference (RNAi)-based genetic screens in embryonic stem (ES), trophectoderm stem (TS) and extraembryonic endoderm (XEN) stem cells.…”

“…13,37 Thus, to be feasible, these bidirectional promoters must possess strong transcriptional activity in a variety of cell types and resist epigenetic silencing commonly associated with stem cell biology and animal transgenics. 13,36,37,43 Here, our goal was to develop a bidirectional lentiviral vector exhibiting strong transcriptional activity with which to conduct RNA interference (RNAi)-based genetic screens in embryonic stem (ES), trophectoderm stem (TS) and extraembryonic endoderm (XEN) stem cells. Short hairpin RNA (shRNA) screens to date have utilized both g-and lenti-class retroviral vectors to deliver RNAi-inducing transgenes.…”

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

“…For random fashion. Multiple copies of the injected construct usually form a concatemer(s) in tandem arrays, in which the copy sequences are often incomplete at the ends, and DNA nibbling is sometimes observed between repeat units (Bishop, 1996;Baup et al, 2010). The repeat number of transgene copies is reported to be unregulated and varies widely between different microinjection experiments within a given host species injected with identical constructs (Würtele et al, 2003).…”

Synthetic design of strong promoters

“…Many of the strong promoters currently in use have highly variable levels of transcription in different cellular contexts and show little or no expression in many cell lineages. This limits the utility of both lossof-function (i.e., shRNA) and gain-of-function (i.e., cDNA expression) genetic screens in many cell types (1), as well as recombinant protein production. Synthetic promoters could conceivably drive transcription in cellular contexts in which current promoter options are not ideal.…”