Lentiviral integration preferences in transgenic mice

Yang, Su–Hua; Cheng, Pei-Hsun; Sullivan, Robert; Thomas, James W.; Chan, Anthony W.S.

doi:10.1002/dvg.20435

Cited by 26 publications

(17 citation statements)

References 41 publications

(84 reference statements)

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“…Concurrent studies documented that lentiviral vectors had been successfully used to generate transgenic mice, rat, pig, cattle, chicken and nonhuman primate [8], [14], [25], [26], [27], [28]. Different transgenic species generated by lentiviral vectors exhibited variability in gene transfer efficiency, transgene expression and epigenetic status.…”

Section: Discussionmentioning

confidence: 99%

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Liu

Wang

et al. 2013

PLoS ONE

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BackgroundLow efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed.Methodology/Principle FindingsEGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep.Conclusions/SignificanceInjection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification.

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Section: Discussionmentioning

confidence: 99%

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Liu

Wang

et al. 2013

PLoS ONE

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“…e ELISA of IL-2 in supernatants of B3Z and MF2.2D9 cells cultured with BM DCs, which were incubated with BL21/pThiOVA for 4 h (n = 3). These experiments were carried out in mice from different FcdGW-FF3 lines, similar results were achieved preferred to integrate into introns in transgenic mice, there were still some integration in exons and other regulator elements, which might influence the development of embryos (Yang et al 2008b). On the other hand, implantation operation is an important factor impacting natalities (Shin et al 2006).…”

Section: Discussionmentioning

confidence: 53%

Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors

et al. 2009

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Dendritic cell (DC) specific transgenic mice are a most important model for investigating dendritic cell functions in vivo. Recently, lentivirus mediated gene transfer has become a powerful and convenient method for generation of transgenic mice. We cloned a 1.2 kb CD11c promoter and constructed a lentiviral vector, which efficiently drove DC-specific expression in vitro. After microinjection of purified virus into the perivitelline space of single-cell embryo, more than 80% newborn mice were transgenic and 7 F0 founders were rapidly generated in 2 months. GFP was strictly expressed in CD11c+ cells in spleens, thymus and lymph nodes of the transgenic mice. Importantly, the physiological characteristics and functions of DCs in the transgenic mice were not altered by the specific expression. These results indicate that this vector could be used to rapidly prepare DC-specific transgenic mice. Thus, this lentiviral vector system may provide a convenient and useful tool to study the properties of DCs in vivo.

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“…The generation of novel vectors including insulator elements to prevent positional-dependent variegation may also represent a possible avenue of investigation to facilitate the establishment of mouse lines with more predictable transgene expression (Pannell and Ellis 2001;Park 2007). However, the unknown integration pattern causing potential interference with endogenous gene expression (Schroder et al 2002;Yang et al 2008) and the possible off-target effects of shRNAs still represent clear limitations to the use of lentiviral transgenesis for the development of reliable loss-of function in vivo mouse models. Fig.…”

Section: Discussionmentioning

confidence: 98%

Variegation and silencing in a lentiviral-based murine transgenic model

et al. 2009

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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.

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Lentiviral integration preferences in transgenic mice

Cited by 26 publications

References 41 publications

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors

Variegation and silencing in a lentiviral-based murine transgenic model

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