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

Abstract

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.

Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis

“…NTC represent the no template control of real-time PCR The current study resulted in all the newborn lambs carrying the GFP gene and 88.9 % of them expressing fluorescence, showing the high efficiency of lentiviral transgenesis. Previous studies reported recently in sheep ranged from 28.6 to 61.5 % of lambs containing the transgene, and in some cases expression was not achieved (Lillico et al 2011;Liu et al 2013;Tian et al 2013). In our case, only one lamb did not show GFP fluorescence under UV light, perhaps due to DNA methylation, a phenomenon that has been reported before acting as a critical factor to regulate transgenic activity (Kong et al 2009;Tian et al 2013).…”

“…This marker allows following the expression of a co-expressed transgene in cells and tissues during normal development or diseases evolution. To date, there are few reports of GFP transgenic sheep produced in the recent years (Deng et al 2013;He et al 2012;Liu et al 2013;Ritchie et al 2009;Tian et al 2013) and more information is required in order to have enough evidence about the effect of the introduction of this reporter gene into the ovine genome.…”

Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats

“…We found that red fluorescence expression in fibroblasts from ear tips was not completely synchronous with that at the phenotypic level. Previous studies have also found differences in the expression patterns of CMV promoter-driven transgenes at the cellular and individual levels in different transgenic animals (Wakula et al, 2011), and tissue specificity has been reported in transgenic cattle (Yang et al, 2008), sheep (Liu et al, 2013), and other heavy livestock. Bisulfite sequencing of the exogenous CMV promoter in 5-Az-treated and untreated transgenic cells revealed a high methylation status in fibroblasts of four of the six transgenic cashmere goats did not exhibit the external red fluorescence phenotype.…”

Production of germline transgenic pigs co-expressing double fluorescent proteins by lentiviral vector

“…Transgenic farm animals (pig, sheep, cattle and quail) produced recently by lentivirus were focused on transferring a single foreign gene to founders (Hofmann et al 2003;Zhang et al 2012;Liu et al 2013;Seidl et al 2013;Xu et al 2013).…”

“…The first lentiviral transgenic pig was produced by injecting lentivirus into zygotes, and had generation rates of between 19 and 33% (Hofmann et al 2003), significantly higher than the 1% obtained by pronuclear DNA microinjection (Hammer et al 1985). Lentiviral vectors could efficiently deliver transgenes into a host cell genome, but almost all of the lentiviral transgenesis studies were confused on the transfer of a single exogenous gene (Lois et al 2002;Hofmann et al 2003;Reichenbach et al 2010;Liu et al 2013). It is difficult to generate multi-transgenic animals using lentiviral technologies.…”