Lambs possessed more than one copy of integrant. The average integrant purchase NHS-Biotin numbers were 3.1 (25/8), ranging from 2 to 7. Depending on literature investigation, the average integrant numbers in transgenic pigs generated by injection of recombinant lentivirus 22948146 were 4.6, ranged from 1 to 20 copies [14]. The variability of integrant numbers was presumably associated with animal species and lentiviral titer injected. Previous investigators had addressed unprecedented high rate of transgene expression [11,12]. To investigate the transgene expression, observation on whole lambs showed green fluorescence in hoof, lip and horn from birth continuously to maturity. Tissues from freshly dead transgenic lambs also presented green fluorescence in liver, kidney and lung. This was consistent with the results reported in pigs and cattles [15,31]. To further verify the expression of transgene, the proteins extracted from different tissues were subjected to western blotting analysis. The GFP protein expression varied among individuals and tissues of transgenic sheep, which was in consistence with the fluorescent intensity observed in vivo fluorescence imaging. In general, the overall expression of transgenic sheep derived from lentiviral transgenesis indicated the wide range of transgene expression in different individuals. However, dramatic disparity of transgene expression was identified in different tissues. Our results were similar to the report in transgenic pigs and birds [19,32]. Variegation of transgene expression might be explained by differences in the basic biology or lentiviral vector activity. On the other hand,these results were obtained from F0 founders. Based on previous report in pig [20], one-third of lentivirusmediated transgenic pigs of F1 generation exhibited low expression levels and hypermethylation. Further studies are worthwhile to carry out to investigate the transgene expression in F1 generation of transgenic sheep in the future. Previous reports showed that DNA methylation has been verified as a critical factor in MedChemExpress (��)-Hexaconazole regulating activity of transgenic vector [23,24,33]. Meanwhile, analysis of methylation status oftransgenic pigs found that the high degree of methylation in the promoter and coding region of lentiviral transgene was accompanied by low levels of transgene expression [20]. To explore the regulation mechanism of lentiviral transgene, we measured CMV promoter methylation levels and analysed the association of promoter methylation with transgene expression in all transgenic founders and part of tissues. Our results showed that the methylation levels ranged from 37.6 to 79.1 in transgenic individuals and 34.7 to 83 in tested tissues. The association of methylation level with GFP expression suggested that GFP expression was inversely correlated with methylation status, both in individuals and in tissues. This result was similar to the outcomes reported in transgenic mice [34] and pigs of somatic cell cloning [33]. The number of lentiviral transgene integrant was reported as an important factor involved in transgene expression besides the methylation [35]. Studies on lentiviral transgenic pigs found that the increase of transgene expression was almost linearly increasing with lentiviral integrant numbers [14]. In this study, there is no any inclination between integrant numbers and GFP expression levels (r = 0.128, p.0.05, datas not shown). We postulated that the lentiviral transgene expression was presumably influenced by in.Lambs possessed more than one copy of integrant. The average integrant numbers were 3.1 (25/8), ranging from 2 to 7. Depending on literature investigation, the average integrant numbers in transgenic pigs generated by injection of recombinant lentivirus 22948146 were 4.6, ranged from 1 to 20 copies [14]. The variability of integrant numbers was presumably associated with animal species and lentiviral titer injected. Previous investigators had addressed unprecedented high rate of transgene expression [11,12]. To investigate the transgene expression, observation on whole lambs showed green fluorescence in hoof, lip and horn from birth continuously to maturity. Tissues from freshly dead transgenic lambs also presented green fluorescence in liver, kidney and lung. This was consistent with the results reported in pigs and cattles [15,31]. To further verify the expression of transgene, the proteins extracted from different tissues were subjected to western blotting analysis. The GFP protein expression varied among individuals and tissues of transgenic sheep, which was in consistence with the fluorescent intensity observed in vivo fluorescence imaging. In general, the overall expression of transgenic sheep derived from lentiviral transgenesis indicated the wide range of transgene expression in different individuals. However, dramatic disparity of transgene expression was identified in different tissues. Our results were similar to the report in transgenic pigs and birds [19,32]. Variegation of transgene expression might be explained by differences in the basic biology or lentiviral vector activity. On the other hand,these results were obtained from F0 founders. Based on previous report in pig [20], one-third of lentivirusmediated transgenic pigs of F1 generation exhibited low expression levels and hypermethylation. Further studies are worthwhile to carry out to investigate the transgene expression in F1 generation of transgenic sheep in the future. Previous reports showed that DNA methylation has been verified as a critical factor in regulating activity of transgenic vector [23,24,33]. Meanwhile, analysis of methylation status oftransgenic
pigs found that the high degree of methylation in the promoter and coding region of lentiviral transgene was accompanied by low levels of transgene expression [20]. To explore the regulation mechanism of lentiviral transgene, we measured CMV promoter methylation levels and analysed the association of promoter methylation with transgene expression in all transgenic founders and part of tissues. Our results showed that the methylation levels ranged from 37.6 to 79.1 in transgenic individuals and 34.7 to 83 in tested tissues. The association of methylation level with GFP expression suggested that GFP expression was inversely correlated with methylation status, both in individuals and in tissues. This result was similar to the outcomes reported in transgenic mice [34] and pigs of somatic cell cloning [33]. The number of lentiviral transgene integrant was reported as an important factor involved in transgene expression besides the methylation [35]. Studies on lentiviral transgenic pigs found that the increase of transgene expression was almost linearly increasing with lentiviral integrant numbers [14]. In this study, there is no any inclination between integrant numbers and GFP expression levels (r = 0.128, p.0.05, datas not shown). We postulated that the lentiviral transgene expression was presumably influenced by in.